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Retrospective Theses and Dissertations 1991 Pathogenesis of respiratory infections caused by Mycoplasma dispar Raul Antonio Almeida Iowa State University Follow this and additional works at: http://lib.dr.iastate.edu/rtd Part of the Animal Sciences Commons, Immunology and Infectious Disease Commons, Medical Immunology Commons, and the Veterinary Medicine Commons Recommended Citation Almeida, Raul Antonio, "Pathogenesis of respiratory infections caused by Mycoplasma dispar " (1991). Retrospective Theses and Dissertations. Paper 9626. This Dissertation is brought to you for free and open access by Digital Repository @ Iowa State University. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Digital Repository @ Iowa State University. For more information, please contact [email protected]. UMI j à MICROFILMED 1992 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 Nortli Zeeb Road. Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 I Order Number 9212128 Pathogenesis of respiratory infections caused by Mycoplasma dispar Almeida, Raul Antonio, Ph.D. Iowa State University, 1991 UMI 300N.ZeebRd. Ann Arbor, MI 48106 Pathogenesis of respiratory infections caused by Mycoplasma dispar by Raul Antonio Almeida A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Microbiology, Immunology, and Preventive Medicine Major: Veterinary Microbiology Approved: Signature was redacted for privacy. In Charge of Major Work Signature was redacted for privacy. FdHhe Major Program Signature was redacted for privacy. Signature was redacted for privacy. ^feâu^Coîié^ Iowa State University Ames, Iowa 1991 L ii TABLE OF CONTENTS Page INTRODUCTION 1 EXPLANATION OF THE DISSERTATION FORMAT 3 LITERATURE REVIEW 4 Defense Mechanisms of the Respiratory Tract Extracellular Mechanisms The Mucociliary Apparatus Response of the mucociliary apparatus to irritants 4 5 6 10 Interaction of pathogenic microorganism with the respiratory mucociliary apparatus 12 Bacteria 12 Viruses 16 Mycoplasmas 19 Cellular Factors 28 Interaction of pathogenic microorganisms with alveolar macrophages 36 Bacteria 36 Viruses 39 Mycoplasmas 42 PART I. "IMPAIRED FUNCTION OF THE MUCOCILIAriY APPARATUS OF CALVES INFECTED WITH Mycoplasma dispar" 48 SUMMARY 50 INTRODUCTION 51 METHODS 53 RESULTS 57 DISCUSSION 68 REFERENCES 73 PART II.- "INTERACTION OF Myœplasma dispar\N\TH BOVINE ALVEOLAR MACROPHAGES" 78 ABSTRACT 80 INTRODUCTION 81 MATERIALS AND METHODS 83 RESULTS 89 DISCUSSION 101 REFERENCES 105 GENERAL CONCLUSIONS 110 REFERENCES 115 ACKNOWLEDGEMENTS 140 1 INTRODUCTION The development of Infectious disease can be understood as the failure of the host's defense mechanism to control proliferation, colonization, and invasion of healthy tissue by pathogenic organisms. Establishment of the infectious process can be the result of the damaging effect of pathogenic microorganisms on cellular elements of the host defense or be enhanced by the host's constitutive defects. Aside from these major determinants, other factors that cause progressive detenoration of the host's defense mechanisms may contribute to the development of disease (Mims, 1982; Roitt, 1988). Stressful situations, deficient nutrition, poor management, and adverse environmental conditions are included among these factors that have been proven to reduce the efficiency of the respiratory defensive mechanisms (Abraham et al., 1986). Microorganisms differ in their capacity to cause disease. Some non pathogenic microorganisms infect the host soon after birth and cannot overcome natural host defenses and they do not produce disease even when the host defense mechanisms are impaired by predisposing factors. Other microorganisms may coexist with the host and they will produce disease only in the event of profound deterioration of the host's defenses. In contrast, highly virulent microorganisms usually produce disease when they infect. Pathogenic microorganisms differ in their persistence in the infected host. Some microorganisms are usually eradicated after the acute disease but other microorganisms may also persist after recovery in healthy carriers; others can lead to dormant infections that may relapse. Still others are present only during active disease. In contrast, infectious microorganisms with moderate virulence. 2 such as some pathogenic mycoplasmas, cause disease characterized by chronicity and long term colonization. The progressively deteriorating condition that results from such infections determines a rupture of the balance between host defenses and microorganism, allowing opportunistic proliferation of microorganisms generally regarded as nonpathogenic. Mycoplasma dispar is a pathogenic mycoplasma species commonly associated with enzootic pneumonia of calves . The infection with this mycoplasma species is characterized by colonization on the surface of respiratory mucous membranes (Gourlay et al., 1970; Thomas et al., 1987). In vitro experiments have indicated that M. d/spar restricts its colonization to the apical surface of respiratory tract ciliated epithelial cells, causing profound alteration of ciliary activity (Thomas et al., 1987). In addition, M. d/spar induced a poor immune response which could help it in evading respiratory defense mechanisms, resulting in long term colonization of these membranes (Howard, 1980; Tanskanen, 1984). The aim of the present research was to characterize the strategy used by M. dispar \o evade respiratory defense mechanisms. The approach used was to determine the effect that M. d/spar infection has on the activity of two major elements of the respiratory defense mechanism of the lungs, the mucociliary apparatus and the alveolar macrophage. Explanation of the Dissertation Format This dissertation was written using the alternate format and consists of an introduction, a literature review, two manuscripts, a general conclusion and a list of references cited in the literature review. The manuscripts were written following the format required by the journal to which these articles will be submitted (American Review of Respiratory Diseases and Infection and Immunity). The remaining sections of this dissertation were written following the format established by the Iowa State University thesis office. The doctoral candidate, Raul Antonio Almeida, is the senior author and principal investigator for each of the manuscripts. LITERATURE REVIEW Defense Mechanisms of the Respiratory Tract The capacity of the respiratory system to eliminate airborne agents is determined by a complex and Interdependent set of extracellular and cellular factors (Phipps, 1981; Dungworth, 1985; Roitt, 1988; Sleigh et al., 1988). Extracellular factors involved in the defense of the respiratory membranes are non-specific and effective as a first defense barrier. These include the mucociliary apparatus, cough, bronchoconstriction reflex, and antibacterial substances present in the secretions that line the airways and alveolar walls (Sleigh et al., 1988). These secretions contain components such as surfactant, bacteriostatic iron-containing compounds, complement fractions, and gamma globulin (Roitt, 1988). Cellular factors involved in the defense of the respiratory tract, include several types of lymphoid cells and their secretory products (German et al., 1990). It was described that the activity of cellular factors was initially nonspecific, but a high degree of specificity was reached soon after the beginning of infectious processes. The achievement of this specific response results from complex and intricate interactions among these cellular elements, which require the synergistic effect of extracellular non-specific factors (Roitt, 1988; Phipps, 1981). The mucociliary apparatus through Its constant neutralization and removal of inhaled infectious and irritant particles constitutes the first and the main defense mechanism of the airways mucosa. In the alveoli, this role is performed by the alveolar macrophage. These cells engulf and inactivate the particulate matter that escapes the mucociliary apparatus, present processed antigens to accesory cells (e.g., B and T lymphocytes), and through the secretion of cytokines, direct the immune response against infectious agents (Sleigh et al., 1988; Khandom et al.,1985; Green et al.,1977) Extracellular Defense Mechanisms Effective removal of harmful agents that are deposited in the respiratory membranes occurs mainly in the upper respiratory tract above the bronchoalveolar junction. Deposition of inhaled particles occurred at different anatomic locations according to their aerodynamic diameter. Those larger than 10 |im are cleared above the larynx and contact with the mucous layer occurred mainly by inertial Impaction (Dungworth, 1985; Sleigh et al., 1988). These large particles impacted against the mucous surfaces of airway passages where air turbulence occurs (i.e., turbinates, airway branching), and the efficiency of such trapping was determined by the size of the particle, by the anatomic complexity of the nasal passages, and by the pattern of respiration (Dungworth, 1985). Particles with aerodynamic diameter between 1 to 3 |im were subject to gravitational settlement. This form of deposition was favored by the relatively still air of deeper parts of the respiratory system (e.g., bronchoalveolar junctions, alveoli). The critical feature of this mechanism, from the point of view of pulmonary homeostasis, was that infectious droplet nuclei and other irritant particles of 1-2 ^m of diameter were more affected than particles of 3 p.m of diameter, and this occurred where the linear velocity of the air stream fell near zero (e.g., the bronchoalveolar junction). In that way, these anatomical regions were exposed to the damaging activity of inhaled infectious agents and irritants (Dungworth, 1985). Deposition of particles with aerodynamic diameters of 0.1 to 0.2 nm occurred by molecular collision which resulted in the settling of these small particles in the alveoli (Dungworth, 1985). Removal of particles from the upper respiratory tract was achieved by the activity of the mucociliary apparatus (McDermott et al., 1982). Small particles which contact the respiratory mucosa at deeper anatomical regions (i.e., bronchoalveolar junction and alveoli) were removed by the combined activity of alveolar macrophages and the mucociliary apparatus (McDermott et al., 1982; Dungworth, 1985). Microorganisms that reach these regions were readily ingested by alveolar macrophages. These cells then leave the alveolar lumen via the tissue and migrate to distant sites through lymphoid follicles, bronchus associated lymphoid tissue (BALT), lymphatics and the blood circulation (Bienenstock etal., 1983; McDermott et al., 1982; Roitt ,1988; Berman et al., 1990) Additionally, it has been described that alveolar macrophages leave the respiratory system via the mucociliary escalator and were ultimately swallowed and destroyed in the gastrointestinal tract (McDermott et al., 1982). The mucociliary apparatus The mucociliary apparatus constitutes one of the most important defenses of the airways against inhaled particles (dust, irritants, bacteria, viruses and allergens) and against changes in the humidity and temperature of the inspired air (Veit and Farrell, 1978; Sleigh et al., 1988). The mucus fraction of this apparatus is secreted onto the mucosal surfaces of the respiratory tract, where it can trap inhaled particles and neutralize irritant gases. This fraction and trapped particles on it are then removed from the airways by coordinated beating of the cilia present on the surface of the respiratory epithelium (Veit and Farrell, 1978; Dungworth, 1985; Sleigh et al., 1988). The mucus fraction of this apparatus was described to be composed of a outer gel and a inner sol fraction (Veit and Farrell, 1978; Sleigh et al., 1988). The trapping of inhaled particles occurred in the outer gel layer and was favored by the stickiness of this fraction. The propelling of this outer fraction and the trapped particles was mediated by the ciliary beating which, in turn, was facilitated by the low viscosity of the inner sol fraction. The viscoelastic and gellike-propieties of the mucus were essential for the transport of trapped particles (Veit and Farrell, 1978; Sleigh et al., 1988). The airway mucus was described as a complex mixture of water, inorganic salts, sulphomucins, sialomucins, neutral mucosubstances, proteins and lipids which were produced and secreted by goblet cells and mucous glands. The inter-and intramolecular cross-linking between these components, mainly through disulphide bonds, largely determined the properties of the mucus (Dungworth, 1985; Phipps, 1981; Sleigh et al., 1988). The mucous surface of the tracheobronchial tree of most mammals was described to be lined by a pseudostratified, ciliated, columnar epithelium which rests on a basal membrane and elastic lamina (Dungworth, 1985; Sleigh et al., 1988). Mucosubstances were locally synthesized and secreted by goblet cells and by serous acini in glands present in the submucosa of cartilage-containing airways (Phipps, 1981 ; Sleigh et al.,1988). The amount of glandular tissue in the submucosa varies among species, ranging from complete absence in birds to one gland per square millimeter in humans (Walsh and McLelland, 1974; Phipps, 1981). When present, these glands accounted for the majority of secretions. Goblet cells have been shown to be irregularly distributed along the respiratory tree. Most of them were in cartilage-containing airways, but some were also found in bronchioles (Thurlbeck et al., 1975; Sleigh et al., 1988). These cells contained large electron-transparent secretory granules composed mainly of acidic glycoproteins (i.e., sulphomucins), although neutral glycoproteins and sialomucins may be also present (Thurlbeck et al., 1975; Sleigh et al., 1988). Epithelial serous cells were found to have a similar pattern of distribution to that described for goblet cells. These cells contained a variable number of electron-dense secretory granules and synthesized neutral glycoproteins, lysozyme and secretory component (Brandtzaeg, 1974; Jeffrey and Reid, 1977; Spiceret al., 1977). Clara cells are another common cell type in the respiratory mucosa (Weibel et al., 1976; Kuhn, 1976; Sleigh et al., 1988). These cells were described as non-ciliated cells presenting a uniform distribution along the respiratory tract. It was also described that the secretory activity of these lipids and glycoprotein-secreting cells responded to environmental conditions (Kuhn, 1976; Phipps, 1981). Lipid secretion might act as a type of bronchiolar surfactant, while glycoprotein secretion could be part of the bronchiolar mucus layer which was described as the beginning of the ainvay mucociliary escalator (Phipps, 1981). In addition, Clara cells were reported to have the central role of stem cells for renewal of other cell types after irritation and damage of the respiratory epithelium (Evans et al., 1978; Kuhn, 1976; Sleigh et al., 1988). The mucosa of the airways was reported to contain various cells types. Among these, ciliated epithelial cells were the most common type. These cells were found down to the terminal bronchioles, increasing their relative number from peripheral to central airways (Breeze and Wheeldon, 1977; Sleigh et al., 1988). On the apical surface of each cell there were up to 250 cilia of 6 |im of length and 0.2-0.3 ^m in diameter, which were anchored to basal bodies located in the cytoplasm of the epithelial cells (Phipps,1981 ; Sleigh et al.,1988). The free portion of each cilia was covered with cell membrane, and inside of the cilia a complex of filamentous contractile elements, composed by nine peripheral double and two central adjacent fibrils was described{Phipps, 1981: Sleigh et al., 1988). This constitutes a highly conserved structure since it was found in all other cilia throughout the animal kingdom (Sleigh, 1977; Sleigh et al., 1988). Claw-like structures have been detected on the tip of the cilia and it was suggested that these structures may play a role in propelling the mucus toward the larynx (Kuhn, 1976; Sleigh et al., 1988). On the apical cell surface of the ciliated cells, a carpet of short non-motile microvilli was described to occur in the spaces between cilia. Mucociliary clearance is performed by the additive effect of an external gel layer of highly viscoelastic mucus that traps inhaled particles and ride on the tip of the cilia, and a internal sol layer of low viscoelastic fluid in which the cilia beat. The propelling effect caused by the cilia beating determines the movement of trapped particles and external mucus toward the larynx. (Veit and Farrell, 1976; Dungworth, 1985; Sleigh et al., 1988). It is not known if these layers represent two separate secretions with independent secretion control mechanisms or if they represent a spontaneous two-phase separation of a polymer mixture (Phipps, 1981 ). As support of the latter concept, it was postulated that the movement of the cilia breaks down the structure of the 10 mucus gel creating a less viscous perclllar fluid (Edwards, 1978). The consistency and depth of this Internal layer determines the efficiency of the propelling activity of the cilia. It was proposed that microvilli of both ciliated and brush cells (an accessory cell type found in low numbers In bronchial and alveolar epithelium) had an absorptive function that helps to control these two features of the perclllar fluid (Jeffrey, 1978; Phlpps, 1981; Sleigh et al., 1988). In addition to the propelling activity, it was described that ciliated cells may also synthesize and secrete sulfated glycoproteins (Sleigh et al., 1988), particularly In response to airway Irritation (Coles, 1987). Response of the mucociliary apparatus to irritants Acute Irritation of the airways with ammonia vapor (100-1000 ppm) or sulfur dioxide (10 ppm) were characterized by a rapid Increase in the secretion of mucosubstances mainly from submucosal glands and from epithelial secretory cells (Richardson et al., 1976; Coles, 1987). It was proposed that this effect was mediated through two pathways which were: A) a reflex neural pathway Involving secretory responses and the stimulation of the cough reflex; and B) a direct action on the mucus secreting cells which Included the release of metabolites of the arachldonic acid pathway (Phlpps, 1981 ; Coles, 1987). The Irritant substances mentioned above also reduced the frequency of the ciliary movement. Thus, the reduced ciliary activity In addition to the decreased viscoelastic properties caused by altered and increased secretions, determined a reduction of the mucus transport (Veit and Farrell, 1978; Sleigh et al., 1988). This effect caused inefficient neutralization and removal of the irritant which resulted in a nflammatory response. Serum also can act as an irritant since it 11 caused stimulation of the secretion of mucin glycoproteins (Phipps, 1981). It was proposed that this effect was mediated through the direct release of luminal surface glycoproteins and increased secretion of the submucosal glands (Phipps,1981). Responses to chronic insult were characterized by increased mucus production and decreased mucociliary function as a consequence of the repeated exposure to irritants or long term infections. These changes were brought about in part, by the loss of cilia and by the hyperplastic reaction of epithelial mucous cells and submucosal glands (Jeffrey and Reid, 1977; Cole, 1987). Changes in the secretory function of the respiratory mucosa were elicited in response to direct and indirect stimulation of the secretory cells. In one study on the effect of cigarette smoke in animals, it was shown that the anti inflammatory agent phenylmethyloxadiazole prevented smoke-induced hypertrophic changes in the epithelium but not in submucosal glands (Jones and Reid, 1978). This was taken as evidence of the direct effect of the irritant on the respiratory epithelium. However, there was also evidence that the metabolically active respiratory mucosa may modulate the function of the underlying smooth muscle by metabolites and the production of relaxant, constrictor, or chemotactic factors (Cuss and Barnes, 1987). The involvement of indirect stimulation in the smoke-induced hypertrophic changes was highlighted in an experiment in which these hypertrophic changes were reproduced by repeated injections of pilocarpine and isoprenaline (Phipps, 1981). In addition, chronic inflammatory processes elicited an increase in the relative number of secretory cells and caused a shift in the composition of secretions of submucosal glands and goblet cells. This resulted in a net increase of the 12 population of cells containing sialomucins (Reid, 1965; Wheeldon et al., 1976). The increased activity of goblet cells and submucosal glands, and the production of secretions of lowered viscosity was coupled with an inability to remove such excess of mucus production by ciliary epithelium that was impaired by the direct damaging effect of the irritant (Jeffrey and Reid, 1977). In addition, this scenario was worsened by the effects of these changes on small airways, which become blocked with mucus, increasing the exposure of the respiratory mucosa to the irritant (Woods et al., 1976; Sleigh et al., 1988). Under these predisposing conditions, bacterial pathogens proliferated and caused disease, such as has been described in human cystic fibrosis. In cystic fibrosis, the basic defect was described as a general dysfunction of exocrine glands which caused changes in the chemical composition of the mucus with alteration of its physical properties (Woods et al., 1976). The mucus of patients with cystic fibrous was reported to contain more mucus glycoproteins, plasma transudate, and DNA than mucus from healthy controls (Woods et al., 1976). It was proposed that physicochemical changes in the mucus may alter the interaction between mucus and cilia, or may alter the bonding between glycoproteins present in the mucus, responsible for their consistency. These changes, in addition to the loss of cilia, lead to impairment of the mucociliary function and to the proliferation of secondary pathogens (Woods et al., 1976). Interaction of pathogenic microorganism with the respiratory mucociliary apparatus Bacteria Pseudomona aeruginosa is the most common cause of morbidity and mortality in cystic fibrosis patients. The bacteria presented tropism 13 for the respiratory tract and adhered to tracheal ciliated cells and tracheobronchial mucins by means of pili or, in mucoid strains, through the bacterial exopolysaccharide (Vishwanath, 1988). The marked tropism and adherence capabilities of P. aeruginosa, in addition to the failure of the host to physically or immunologically remove the bacteria from colonized cells, determined the persistence of the infection and the non-favorable outcome of the disease (Rampai, 1987; Vishwanath, 1988). Pseudomona aeruginosa was also reported to induce ciliary and epithelial necrosis, which worsen even more the prognosis of cystic fibrosis. Similarly, Haemophilus influenzae caused dyskinesia and destruction of the ciliary epithelium. Encapsulated H. influenzae strains were shown to reduce the ciliary activity more than the unencapsulated variant of the bacteria (Cole, 1987; Reynolds, 1987). Several members of the Bordetella genus also exhibited a marked affinity for the ciliated epithelium. Of the various toxins and virulence-related factors produced by Bordetella pertussis, only one has been demonstrated to reproduce the specific respiratory epithelial cytopathologic changes characteristic of the pertussis syndrome (Goldman and Herwaldt, 1985). This factor, known as tracheal cytotoxin (TCT), was released by B. pertussis during log phase growth. Intratracheal exposure to purified TCT specifically damages ciliated epithelial cells, causing ciliostasis and extrusion of these cells (Goldman and Henwaldt, 1985). The particular disaccharide-tetrapeptide structure of TCT reveals that it was apparently formed by cleavage of peptidoglycan (Goldman and Cookson, 1988). Unlike other gram-negative bacteria, B. pertussis appears to be very selective in the release of cell wall fragments, since more than 95% of soluble peptidoglycan in culture 14 supernatants was TCT. The structure of TCT places it in the "muramyl peptide" family, a group of structurally related molecules that were responsible for a diverse array of biological activities (Goldman and Cookson, 1988). The selective biological activity of B. pertussis TCT was studied in tracheal organ cultures by light and electron microscopy. From this study it was reported that TCT only affected ciliated cells, and these underwent a series of pathological changes that preceded their eventual extrusion (Goldman and Herwaldt, 1985). In addition to TCT, two other proteins secreted by B. pertussis mediated the adherence of this or other bacterial pathogens to mammalian respiratory cilia (Tuomanen, 1986). When either ciliated cells or other pathogenic bacteria, such as Streptococcus pneumoniae, H. influenzae, and Staphylococcus aureus were treated with these adhesins, they acquired the ability to adhere to cilia in vitro or in vivo. Such piracy of adhesins may contribute to superinfection of mucosal surfaces by secondary pathogens as is commonly seen in respiratory diseases such as whooping cough (Tuomanen, 1986). Muramyl-peptide molecules (one of which is identical to TCT) were described as being released by Neisseria gonorrhoeae. This compund can cause ciliated cell-specific damage like that described during gonococcal infection of fallopian tube mucosa (Goldman and Cookson, 1988). Inoculation of lambs with Bordetella parapertussis also caused alteration in the mucociliary apparatus (Chen et al., 1988). In the first 24 hours after infection, tracheal and bronchial epithelium was covered by a large amount of inflammatory exudate. Later, the tracheobronchial epithelium showed focal extrusions of ciliated cells, which were occasionally associated with B. parapertussis organisms (Chen et al., 1988). Cytopathological changes F 16 associated with B. parapertussis infection were mild focal degeneration of airway epithelium with slight loss of cilia, and focal inflammation of the lungs with moderate to severe degeneration of type I and type II pneumocytes. In addition to these cells, alveolar macrophages presented pathological changes which included moderate to severe cytoplasmic vacuolization (Chen et al., 1988). Bordetella bronchiseptica, has been described as one of the etiological agents of atrophic rhinitis in pigs and "kennel pneumonia" in dogs (Dungworth, 1985). This bacterium showed tropism for the ciliated epithelium of the respiratory tract and possesed readily detectable somatic pili. This was the only consistent phenotypic characteristic associated with the ability to induce ciliostasis (Bemis and Wilson, 1985). Filiated strains of B. bronchiseptica treated with proteases lost their capacity to attach to cilia and the capacity to induce ciliostasis (Bemis and Wilson, 1985). In another work, ciliated epithelial cell outgrowths from canine tracheal expiants were used to study the interaction between B. bronchiseptica and respiratory cilia (Bemis and Kennedy, 1981). Phase I and intermediate-phase B. bronchiseptica isolates caused reductions (more than or equal to 50%) in ciliary beat frequencies within 5 minutes of infection, and nearly complete ciliostasis within 3 hours after infection. Rough B. bronchiseptica and heat and formalin-killed preparations of the phase I isolate had no ciliostatic effect. Phase I and intermediate-phase isolates attached to cilia, whereas the rough variant did not. The ciliostatic effects of the phase I isolate could not be reproduced with endotoxin or culture supernatants from the organism. From this experiment it was concluded that attachment alone does not produce ciliostasis but mediation of ciliostatic effects of 16 Bordetella may require the close association between metabolically active organisms and cilia (Bemis and Kennedy, 1981). The avian pathogen Bordetella avium also exhibited affinity for the ciliated epithelium. In one study, one-day-old turkeys were infected intranasally with B. avium and tracheas were examined by scanning and transmission electron microscopy 1 to 5 weeks post-inoculation. From these it was described that infection with this pathogen caused progressive loss of ciliated epithelium with replacement by non-ciliated cells. In addition, bacterial colonization of ciliated cells was associated with membrane-bound crystalline inclusions in the cytoplasm of epithelial cells, depletion of secretory granules, and distortion of tracheal rings and of the mucosal surface (Fagerland and Arp, 1987). Viruses Bovine herpes virus-1 (BHV-1) caused profound alterations of the respiratory epithelium of the upper regions of the respiratory tract upon infection. In experimental cases, rhinitis, laryngitis and tracheitis developed with pinpoint haemorrhages on the mucosal surface 4, 5 or 7 days after infection (Allan and Msolla, 1980). There was neutrophilic infiltration of the hypertrophied epithelium with extensive loss of cilia, leaving areas of epithelium covered by microvilli (Allan and Msolla, 1980). The submucosa of these areas was infiltrated with lymphocytes and the tracheal submucosal glands were dilated and slightly hypertrophied. Lesions were of similar severity on day 5 and 7 after infection and virus was re-isolated from nasal passages from days 412 after infection (Allan and Msolla, 1980). The importance of the infection by this viral agent in the development of more severe respiratory processes was stressed by Jericho (1983). This author described that the main histopathologic 17 changes observed in a mixed infection with BHV-1 and P. hemolytica were hyperemia of pharyngeal tonsils and the lamina propria of air passages and alveolitis associated with edema and fibrin deposition in lumen and septa. Initially, eosinophilic granulocytes were numerous in turbinates, bronchi and lung, but 3 days after exposure, polymorphonuclear leukocytes predominated. Bacterial colonies were seen in nasal turbinates, tonsils, trachea and lung. Necrosis was seen in the nasal, tracheal, and pulmonary epithelium. Necropurulent changes were particularly evident in tonsils and alveoli. Blood vessels, walls of bronchioles, and major air passages were generally free from inflammation (Jericho, 1983). Parainfluenza-3 virus (PI-3) was also described to induce damage to the respiratory epithelium. In the early acute stage of the pneumonia, PI-3 replication was observed within epithelial cells of the respiratory tract and within alveolar macrophages (Bryson et al., 1983). Morphological changes included loss of cilia and of ciliated cells in small bronchi and bronchioles. Necrosis was noted in both bronchiolar and alveolar epithelium. Proliferation of non-ciliated bronchiolar cells (Clara cells) produced zones of hyperplastic bronchiolar epithelium. Within the alveoli, widespread proliferation of type 2 pneumonocytes led to alveolar epithelization. Organization of bronchiolar exudate involving fibroblasts and macrophages was widespread in calves killed between 7 and 12 days after Infection and in calves killed at 12 days and subsequently, bronchiolitis obliterans was widespread ( Bryson et al., 1983). Moreover, in another study, electron microscopic demonstration of aggregates of viral nucleocapsids in the cytoplasm, alterations of cilia and basal bodies, dissolution of cytoplasmic membranes, and shedding of virus into luminal 18 spaces confirmed that epithelial cells of the respiratory tract were the primary target cells for PI-3 replication (Tsai and Thompson, 1975). These observations revealed that productive infection was more efficient in the bronchioalveolar junctions and destructive changes were more pronounced in the tracheal epithelium. The finding that PI-3 virus replicates in alveolar type II cells, which are considered to be the producers of pulmonary surfactant, suggests that changes in surfactant production may occur during the peak of infection of these cells (Tsai and Thompson, 1975). The demonstration of virus penetration through the basolateral aspect of epithelial cells in small bronchioles and the presence of virus particles in the interstitial regions imply that host defense barriers may be impaired by virus invasion. Bovine respiratory syncytial virus (BRSV) also had a marked tropism for ciliated respiratory epithelium (Castleman et al., 1985). In experimental infections of calves with BRSV, there was hyperplasia of basal epithelial cells in association with other epithelial morphological changes . These lesions resulted in the loss of normal ciliated epithelium in these airways 5 to 7 days after infection (Castleman et al., 1985). Regeneration of airway epithelium was largely completed by 10 days after inoculation, except in 1 of 4 calves that developed secondary bacterial pneumonia. The pulmonary ultrastructure of BRSV inoculated calves 30 days after inoculation was indistinguishable from that of controls. These results demonstrated that BRSV can induce reversible alterations in airway epithelium, which may cause depression of mucociliary clearance and thereby enhance susceptibility to bacterial infection (Castleman et al., 1985). In another work, 33 cattle with fatal respiratory tract diseases were examined for gross and histopathologic lesions and presence of viral and 19 bacterial pathogens in the lungs. Fifteen animals had lesions that were classified as atypical interstitial pneumonia ( AlP), and the histologic features of these included interstitial edema, thickening of alveolar walls, hyaline membrane formation and hyperplasia of type -II pneumonocytes (Collins et al., 1988). Bovine respiratory syncytial virus was detected in 11 of these 15 cattle with AlP. The significant association found between these lesions and the presence of BRSV led these researchers to postulate BRSV as an etiologic factor of AlP (Collins et al., 1988). Mvcoplasmas Several species of Mycoplasmas can produce alteration of the ciliated respiratory epithelium of humans or domestic animals. These alterations were observed under in vitro conditions, using tracheal expiants, or under in vivo conditions. For instance, Jones et al. (1985) using tracheal expiants from fetal lambs or newborn kids showed that Mycoplasma ovipneumoniae and Mycoplasma arginini were capable of stopping ciliary beating and causing the loss of these appendages. Direct observation using scanning electron microscopy showed clusters of microorganisms attached to epithelial surfaces and in association with cilia (Jones et al., 1985). In calves naturally infected with M. dispar and Ureaplasma diversum, the respiratory tract presented alterations of the mucociliary escalator (Allan et al., 1977). Among these, morphological changes of the bronchial epithelial cells included hyperplasia, loss of cilia, cytoplasmic protrusion into the airway lumen, and distended and dismpted mitochondria (Allan et al., 1977). In addition, changes in the composition of the mucus were reported, including a net increase of sialomucin production. In later stages of the infection, some of the 20 cells lost their characteristic histochemical staining due to exhaustion or replacement by a different or immature cell type (Allan et al., 1977). Using tracheal organ cultures, it was shown that M. dispar was capable of stopping ciliary motility and destroying the ciliated epithelium (Thomas and Howard, 1974; Howard and Thomas, 1974; Thomas et al., 1987). In these studies it was observed that the inhibitory activity on ciliary motility caused by M. dispar was unaffected by catalase, ruling out the production of H2O2 as mediator of this effect. It was shown, however, that the inhibitory effect on the cilia was dependent on the concentration of fetal bovine serum (FBS) in the culture medium (Howard and Thomas, 1974), In a comparative study of the growth rate of M. dispar m serum-free media or with the addition of 5 % of FBS, these authors reported that in spite of the similar final mycoplasma titer observed under both conditions, faster growth of M. dispar was observed when serum was added, compared with that observed in the serum-free condition. Based on this observation, it was postulated that a toxic substance could be synthesized and released into the medium, and this putative molecule could be derived either from FBS or produced at high concentration in the presence of FBS. However, no changes were observed when filtered supernatants from M. dispar infected cultures were used. (Howard and Thomas, 1974). The effect of porcine mycoplasmas on the ciliated respiratory epithelium was studied using tracheal organ cultures (Pijoan et al., 1972). In this work, it was shown that Mycoplasma granularum, Mycoplasma hyorhirjis and Mycoplasma hyosynoviae were able to cause more than 50 % cilia-stopping effect (CSE) after 7 days of inoculation. In contrast, in the same system Mycoplasma hyopneumoniae caused CSE that was below or slightly above the 21 effect observed in the non-inoculated controls. The titer of these porcine mycoplasmas in the pig tracheal organ cultures used in this study remained constant at levels of IQS-IO? color changing units/ml (ccu/ml) in these mycoplasmas with the exception of M. hyopneumoniae. This indicated that the CSE exerted by the latter mycoplasma species was dependent on the titer of the microorganism. This observation was confirmed in the work of Williams and Gallagher (1977), in which these researchers, using porcine tracheal rings supplemented with monolayer cultures of fetal lung fibroblasts, were able to demonstrate severe cellular damage and CSE in tracheal rings infected with M. hyopneumoniae. Using this organ culture system, the M. hyopneumoniae titer increased from 101 to 10® ccu/ml after 3 days of inoculation (Williams and Gallagher, 1977). Using chicken tracheal cultures. Cherry and Taylor-Robinson (1970) showed that Mycoplasma galiisepticum was capable of stopping ciliary activity and that the effect was not related with the infecting dose. It was also shown that the addition of neuraminidase or catalase to the cultures did not inhibit the CSE. These observations were taken as evidence to postulate that neither cytoadsorption nor peroxide production were important factors in the virulence of this mycoplasma. However, these authors reported the presence of molecule(s) toxic for the ciliated epithelium (Cherry and Taylor-Robinson, 1970b). In contrast, the same authors using a similar model, reported that the CSE exerted by Mycoplasma mycoides Msx.capri was directly associated with the number of organism inoculated. The observed CSE was dependent upon a close association of multiplying mycoplasma, and the addition of catalase or glucose protected the cilia from such effect. No demonstrable toxic factors were 22 related with these changes in the ciliary epithelium (Cherry and TaylorRobinson, 1970a). Mycoplasma pneumoniae is a pathogen of the human respiratory tract. This mycoplasma species was described as the principal cause of atypical pneumonia in children and young adults (Denny et al., 1971; Foy et al., 1979). Clinical manifestations of the disease range from asymptomatic conditions to bronchitis and pneumonia. The damage originated by M. pneumoniae infection has been studied using tracheal organ cultures and the changes detected included gradual loss of the ciliary activity, nuclear swelling, cytoplasmic vacuolization and exfoliation of ciliated cells (Carson et al., 1980). These changes were accompanied by metabolic abnormalities, which included decreases in nucleotide content, intracellular levels of ATP, carbohydrate utilization, amino acid transport, macromolecular synthesis, and oxidative metabolism (Gabridge and Polisky, 1977; Upchurch and Gabridge,1982; Hu et al., 1976; Gabridge, 1975). In another study, chicken-embryo tracheal organ cultures were inoculated with M. arginini, M. equigenitalium, 2 strains of M. subdolum, Acholeplasma laidlawii and 3 strains of A. oculi (Moorthy and Spradbrow, 1985). All these strains established and multiplied in the expiant cultures, but only M. subdolum and A. oculi produced a cytopathic effect on ciliated epithelial cells, that ended with the sloughing of these cells after 6 days. There was a positive correlation between ciliostasis and increased in the titer of both M. subdolum and A. oculi, but such a relationship was not detected with M. equigenitalium and A. laidlawii (Moorthy and Spradbrow, 1985). Cells infected with M. subdolum showed sloughing of cilia, vacuolization, and increase in size of mitochondria, followed by disorganization of epithelium and marked destruction of cellular organelles (Moorthy and Spradbrow, 1985). Mycoplasmas were attached to the epithelial surface of the tracheal expiant 8 days after infection (Moorthy and Spradbrow, 1985). Even when these results indicated that these equine mycoplasma species can induce cytopathic effects on chick embryo tracheal expiants, the same can not be taken as valid for equine ciliated respiratory epithelium. Using a similar in vitro approach but complemented with in vivo observations, Dykstra et al. (1985) studied the pathological effect induced by M. gallisepticum on ciliated respiratory epithelium of chickens. These workers described that the changes in the tracheal epithelial surfaces induced by the mycoplasma infection under in vivo and in vitro conditions, included release of granules containing mucus, followed by exfoliation of ciliated and non-ciliated epithelial cells. Loss of cilia from individual cells was infrequent, but in those cells in which this change was detected, this was followed by loss of the epithelial cell's intercellular connections. These events were then followed by rounding up and exfoliation of cells with degradation of their membranes (Dystkra et al., 1985). Thus, this process gave rise to a population of released cellular organelles, such as mitochondria, which along with the sloughed cilia and mucus formed the exudate present within the tracheal lumen (Dystkra et al., 1985). Repair of the epithelial surface was effected by basal epithelial cells that began to differentiate and fill the spaces left by exfoliated cells. Fourteen days after infection, these cells were hypertrophic and non-ciliated but covered with microvilli (Dystkra et al., 1985). Histologically, there was increased epithelial thickness due to cellular infiltration and edema. Results from experiments with tracheal rings showed similar changes, with the exception that exfoliation of the 24 epithelial cells occurred sooner after infection and was characterized by more severe ultrastuctural changes than that observed in vivo (Dystkra et al., 1985). Finally, using a similar in vitro model, it was shown that two strains of M. synoviae and one strain of M. gallisepticum exerted pathological effects on the ciliary respiratory epithelium of chickens (Hirano et.al., 1976). Mycoplasma gallisepticum was more effective in stopping ciliary activity than the two M. synoviae strains, and electron microscopic examination showed the mycoplasma attached to cilia and epithelial cells (Hirano et.al., 1976). Mycoplasma hyorhinis was shown to produce alterations on the ciliated epithelium under in vivo conditions (Baskerville and Wright, 1973). Twenty-four 4-week-old pigs were infected intranasally with M. hyorhinis isolated from lungs of animals with enzootic pneumonia. All the animals that were slaughtered between 15 days to 8 weeks after infection showed widespread pulmonary consolidation (Baskerville and Wright, 1973). Electron microscopic examinations revealed mycoplasmas in the lumen of airways. Ultrastructural changes included large masses of cytoplasm projected from the apex of many ciliated bronchiolar cells, and in these, cilia lost their upright position, became matted, and appeared intertwined (Baskerville and Wright, 1973). The affinity of mycoplasmas for ciliated stmctures is well documented. Mycoplasmas not only show affinity to respiratory ciliated cells, but also to similar stmctures present in other anatomical locations. Scanning electronmicroscopic observation of the mucous membrane of an infraorbital turkey sinus infected with M. gallisepticum revealed a swollen corrugated membrane surface and swollen dome-shaped epithelial cells. All cilia were lost, although microvilli remained. No necrosis of mucosal surfaces was detected (Sanger, 1973). In another study, gnotobiotic mice were inoculated intranasally with M. pulmonis (Haliiwell et al., 1974). Lesions were detected in the middle ear, and among these, proliferation of mucus-secreting cells in the medial wall of the timpanic cavity and areas of ciliary fusion and ciliary loss were described. It was noted that increased numbers of mononuclear cells were accompanied by edema and proliferation of submucosal connective tissue (Haliiwell et al., 1974). Ureaplasma species isolated from the human genital tract and from the genital and respiratory tract of cattle stopped the ciliary activity of the ciliated genital epithelium when cultured in organ cultures of bovine oviduct (Stalheim et al., 1976). This effect was noticed between 24 and 144 hours after inoculation and cellular changes detected by SEM were the collapse and sloughing of cila. In addition, vacuolization, disorganization, necrosis and sloughing of the ciliated epithelium was detected. The mycoplasmal tropism for ciliated cells was well documented by Kohn et al. (1977). In this report, these authors communicated the induction of hydrocephalus as consequence of intracerebral inoculation of M. pulmonis. This congenital teratogenic defect was induced in 116 of 120 rats (96%) and in 23 of 28 hamsters (82%). Electron microscopic examinations revealed that an amorphous material covered portions of the ependymal surface and fusion of the cilia on ciliated cells was also seen. It was suggested that the hydrocephalus was due to ciliary dysfunction or to an imbalance of cerebrospinal fluid secretion and absorption (Kohn et al., 1977). In another work porcine lateral ventricular ependymal cells were used to assert the effect of M. hyopneumoniae and M. hyorhinis on the central nervous system of pigs. It was reported that these mycoplasma species caused degenerative processes which included loss of microvilli and cilia. 26 membrane disruption, and colonization of these ciliated cells by mycoplasmas (Williams and Gallagher, 1981). It was previously shown that ciliary structure was highly conserved throughout the animal kingdom (Sleigh, 1977). It Is possible that the tropism shown by several species of mycoplasma to ciliated cells, reflects the affinity of these microorganisms to a common ciliary surface structure. The attachment of mycoplasmas to host cells was described as a prerequisite for colonization and induction of pathogenic changes in these cells (Hansen et al., 1979; Razin, 1978; Lipman et al., 1969; Sobeslavsky et al., 1968). Flask- shape mycoplasmas (e.g., M. pneumoniae and M. gallisepticum) have been described to possess protein receptors localized in the tip of these microorganisms which specifically interact with neuraminidase-sensitive receptors in host cell membranes (Bredt et al., 1981). It was shown that M. pneumoniae cells interact through their receptor with sialo-oligosaccharide branched structures (sialosyl-l and sialosyl-i) as well as with other related oligosaccharide structures of poly-A/acetylglucosamine type. In the respiratory mucosa, these receptors were only expressed in ciliated cells (Loveless and Feizi, 1989). Long-chain oligosaccharides were found highly polarized at the luminal aspects of the ciliated cells, where the branched structures (sialosyl-l and sialosyl-i) were detected both at the epithelial apical border and on the cilia (Loveless and Feizi, 1989). The distribution and the density of these receptors on the luminal surface of the ciliated epithelium may explain the close association of this mycoplasma species with the cilia and the apical surface of the ciliated cells (Hu et al., 1977). In addition, it was shown that M. pneumoniae could rearrange its shape and increase the amount of binding sites on the specialized tip upon proximity to target cells (Kahane, 1984; Kahane et al., 1984). These changes provide the mycoplasma with an advantageous shape and surface receptor distribution to pass between cilia and microvilli, reaching the membrane surface of ciliated respiratory cells where this mycoplasma attaches (Hu et al., 1977). In contrast, other non-flask shaped mycoplasma which affect the respiratory tract (e.g., M. pulmonis, M. dispar, M. hyopneumoniae), did not show these binding sites on their membrane surfaces, nor specialized tips or ability to rearrange surface receptor density and shape, which can explain the fact that they were only seen in close association with the microvilli present on the apical surface of the ciliated respiratory cells (Baskerville and Wright, 1973; Allan, 1978; Razin, 1978; Kirchhoff, et al., 1984; Kahane, 1984). There appear to be several possible mechanisms that contribute to host cell dysfunction during mycoplasma infection. One of these was the production of hydrogen peroxide and superoxide with oxidative damage and alteration of the redox potential, which was supported by the data reported by Almagor et al. (1983; 1986; 1984), and by the work of Cherry and Taylor Robinson (1970a). Host cell membrane alteration induced by mycoplasma membrane components was reported and proposed as a pathogenic mechanism and production of toxic compounds with CSE was described in M. gallisepticum infection of tracheal expiants (Cherry and Taylor-Robison,1970a;1970b; Gabridge et al., 1974). Finally, the drainage of nutrients from the cell caused by the mycoplasma parasitism was proposed and supported by the protective effect of adenine sulfate in the prevention of cytopathic effects and CSE caused by M. pneumoniae (Gabridge and Garden Stahl, 1978). It is possible that the combination of all these and not the isolated effect of an individual factor are 28 likely to contribute to the induction of pathogenic effects detected on the ciliated epithelium infected with mycoplasmas. Cellular Factors The respiratory tract, as well as skin and the gastrointestinal tract, can be considered as an interface between the sterile internal sanctuary of the body and the environment. It would be advantageous to have, in addition to a non specific clearing mechanism, an antigen-responsive cellular component in the lung that serves as a first-line defense as well as for local antigen sampling. Such cellular factors could allow the adjustment and fine tuning of the specificity of the immune response to antigens present in the environment. This function is fulfilled by the lung lymphoid tissue and the alveolar macrophage. Normally, the lung parenchima contains lymphocytes at defined sites in the lung. Among these sites are the bronchus-associated lymphoid tissue (BALT), lymphoreticular aggregates, and the hilar and paratracheal lymph nodes. There were also a small number of lymphocytes residing within the lung interstitium (German et al., 1990). The relative number of lymphocytes increases upon the exposure of the lung to chemoattractants released by the presence of antigens, and the entrance of these cells to the infected parenchima is not a random process but rather involves a series of defined steps. These included adherence of blood lymphocytes to the vascular endothelium and subsequent directed extravasation and ameboid motion through the tissue in response to locally produced chemotactic factors (Freitas and Bognacki, 1979; German et al., 1990). The exit of lymphocytes from blood to organized lymphoid tissue occurs F 29 at the post-capillary venules, known as high endothelial venules (HEV) (Stamper and Woodmff, 1976). The venular endothelial cells were described as having a plump shape protruding into the vessel lumen, in contrast to normal flattened endothelium (Stamper and Woodruff, 1976). Under non-inflammatory conditions, high endothelial venules were usually observed in lymphoid tissue, but these structures were widely found in tissue undergoing inflammatory processes with lymphocytic infiltration (Freemont, 1983). Gamma interferon (ylFN) appears to possess a dual inducer role of HEV and surface adhesive molecules on endothelial cells (Diuvestijn et al., 1986; Diuvestijn and Hamann, 1989). Adherence of lymphocytes to HEV was described to be mediated by the interaction of specialized vascular addressins on HEV which included intercellular adhesion molecule-1 (ICAM-1) and class II major histocompatibilty complex (MHC) antigens such as HLA-DR (Diuvestijn and Hamann, 1989; Hamann et al., 1988; Haskard et al., 1986). Neither ICAM-1 nor class II MHC antigens were present in normal vascular endothelium, but appeared to be induced by treatment with inflammatory cytokines, chiefly ylFN (Haskard et al., 1986; Masuyama et al., 1986; Pober, 1986). Endothelial cells stimulated with ylFN have been described as "activated", and under this state they were capable of expressing adherence molecules and synthetizing cytokines (German et al., 1990). Thus, local secretion of cytokines induced during inflammation can alter the traffic through the endothelium by regulating the presence of lymphocyte adherence molecules (Curtis et al., 1988; German et al., 1990). After adherence, translocation of lymphocytes occurred through ameboid movements. This involved changes in the cytoskeleton, particularly in actin filaments and microtubules which, in turn, were originated from the interaction of a chemoattractant with its receptor in the membrane surface of the target cell (German et al., 1990). As a result of this interaction, biochemical signals were generated that resulted in not only changes in the cytoskeleton but also induced lymphocytes to enter into an "activated" state that included the progression of the cell from Go to Gi, increased synthesis and secretion of chemoattractants, and increased expression of surface receptors, including lymphocyte function-associated antigen-1 (LFA-1) and CD-4 (Allen et al., 1988; German et al., 1990). Organized mucosal lymphoid follicles in close apposition to the bronchial epithelium have been noted for over a hundred years. These lymphoid aggregates were named BALT by Bienenstock et al. (1973), due to the similitude with the gut-associated lymphoid tissue (GALT). Studies on the distribution of BALT in different species, indicated that rats, rabbits, and guinea pigs showed the most extensive distribution of these accumulations, whereas its presence has been disputed in humans (German et al., 1990). The BALT was described to be absent at birth, developing later at sites of particle deposition in response to postnatal antigen exposure (Plesch, 1983). The main characteristic of BALT was the presence of overlying lymphoepithelium in which epithelial cells were attenuated and flattened and ciliated cells were absent. This lymphoepithelium allowed the passage of soluble and particulate material from the bronchial lumen to the underlying lymphoid tissue (Berman et al., 1990). Under this modified epithelium, a meshwork of lymphocytes and cells that express class II MHC antigens were present. The class II antigen-expressing cells were macrophage-like antigen-processing and-presenting cells needed to activated T cells (Simecka et al., 1986). This lymphoepithelial network had been shown to contain specialized capillary endothelial cells that facilitate the transit of lymphocytes from the blood to BALT (Bermann et al., 1990). Both B and T lymphocytes were found In BALT. In contrast with lymphonodules, no T cell-dependent areas were found in BALT. However, there were areas that predominantly contained B or T cells. (Breel et al., 1988). Studies performed in young rats and in rabbits, indicated the propensity of B cells to express IgA, but as the animal aged, the numbers of these cells decreased In comparison with IgM- and IgG-bearing cells. Nonetheless, BALT together with GALT was though to be a major source of IgA producing B cells (Rudzik et al., 1975). Most of the cells types that were found in BALT, including lymphoepithelial cells, expressed class II MHC antigens (Simecka, 1986). The presence of this structure was in concordance with the proposed function of BALT as a local response site for antigen introduced with the inhaled air (Berman et al., 1990). The proposed theory was that inhaled antigens can percolate through the lympoephitelium and be taken up and be processed by class II MHC-expressing antigen processing cells. These cells then would be capable of presenting processed antigens to helper T-cells which could provide the necessary stimuli for humoral and cytotoxic responses to such foreign molecules (Bermann et al., 1990). There was evidence of traffic of antigen-activated lymphocytes through the lymph to blood and hence, to other organized lymphoid tissue. Thus, cell clones expanded through contact with inhaled antigen might recirculate preferentially to mucosal sites, including GALT, BALT, salivary and cervical mucosa (Bienenstock et al., 1983). Eighty percent of the cells present in bronchoalveolar lavage (BAL) were macrophages and 5 to 10 % were lymphocytes. From the latter, approximately 60% were T cells and 10% B-cells. This leaves as much as 40% of the lymphocytes in BAL unaccounted for as "null cells" (Hunninghake et al., 1979; Young et al., 1984). Lymphocytes showing the phenotype of natural killer cells (NK) were found in normal lungs and this organ has been shown to be a major source of NK activity. In this regard, it was shown that NK-cells derived from bone marrow precursors and they matured in major NK reservoirs such as spleen and lungs. This suggested that precursors were recruited to and matured in the lung much like tissue macrophages (Bender et al., 1987). Lymphocytes obtained from BAL were shown to possess functional activity of every type, including immunoglobulin production, secretion of lymphokines, cytotoxicity, expression and activation of adhesion molecules, and NK function (Berman et al., 1990). However, studies of lymphocytes from normaJ lungs provided the view that these cells were hyporeactive in NK activity and proliferative and antibody production responses to mitogens when compared with blood-derived lymphocytes (Robinson et al., 1984; Kaltreider and Salmon, 1973). In this regard, it was found that surfactant lipids have a profound suppressive effect on lymphocyte proliferation and effector cell function (Shimizu et al., 1988; Wilsher et al., 1988). In regards to T cells, it was found that 8 to 20% of the CD3+ cells in the lung, expressed yS T cell receptor. This type of T cell receptor was also described to be present in high numbers in intraepithelial lymphocytes of the intestine and dendritic cells of the skin, and cells showing this surface antigen were also highly represented in responses following the administration of protein purified derivative (Augustin et al., 1989). These results suggested the presence of a lung-specific population of T cells (Berman, 1990). It was established that alveolar macrophages were unique among other macrophages. They differ in their restricted residence in the lung and by their metabolism particularly adapted to the aerobic conditions of the lung alveoli (Khandom et al., 1985; Henson et al., 1984). Alveolar macrophages were described as the principal free resident cells of the distal airspaces and responsible for maintaining a microorganism-free environment in the lower respiratory tract (Glasgow et al., 1989). In addition, this cell was thought to play a role in the mediation of acute lung injury (Brieland et al., 1987), as well as in resolving pulmonary inflammation (Dal Nogare and Towens, 1990). The initial inflammatory events originated as a result of the proliferation of pathogenic microorganisms in the lower regions of the respiratory tract were characterized by increased vascular permeability and accumulation of inflammatory cells, which at an early step (6 hours) were leukocyte polymorphonuclear cells (PMN) (Henson, 1984; German et al., 1990). By 48 hours and beginning as early as 6 hours after the initiation of the inflammatory changes, the cellular infiltrate has shifted to lymphocytes and mononuclear monocytes (phagocytes). By day 7 to 10, in uncomplicated cases, the lesion was resolved and a solid immune response was established (Henson, 1984). This final state was achieved by the interplay of macrophages, lymphocytes and cytokines produced and secreted by these cells types (Berman et al., 1990). After the initial increase of vascular permeability and migration of PMNs, probably elicited by the complement fractions Cab and Cga, the activity of macrophages was characterized by engulfment and processing of antigens. Concomitantly, macrophages secreted interleukin 1 (IL-1) which activated T cells that migrated to the foci of inflammation (Roitt, 1988). Activated T cells produced interlukin 2 (IL-2) which activated additional T-cells, amplifying the T cell response. Activated T- cells also secreted interleukin 4 (B-cell stimulatory factor) interleukin 6 (8 cell growth factor), and B cell differentiation factors which, synergistically, caused the activation and maturation of B cells responsible for the humoral phase of the immune reaction (Roitt, 1988). In addition, activated T helper cells secreted macrophage chemotactic factors, macrophage migration inhibition factors, and macrophage activating factors which mediated the activation and recruitment of monocytes from the blood circulation to the foci of inflammation (Roitt, 1988; Fuchs et al., 1989). Even when most of the cellular amplification and recruitment was mediated via T helper cells and their interleukins, the triggering factor (IL-1) is launched by alveolar macrophages. It was described that monocyte cells recruited from the blood undergo an activation process during which these cells developed increased functional capabilities compared to those of the alveolar macrophages. These activated monocytes, through an increased capacity of synthesis and secretion of IL-1, originated a positive feed-back mechanism, which amplified the inflammatory response even more (Fuchs et al., 1989). These activated macrophages were "armed" with receptors that mediated the removal of aged PMNs and cellular debris from the area of inflammation (Garret et al., 1984, Henson et al., 1984, Dal Nogare and Towens, 1990). These activated cells presented increased capabilities for the production and secretions of factors needed for the healing and the repair of the lung lesions. In this sense, recruited monocytes differ from resident macrophages (Dal Nogare and Towens, 1990). Recruited macrophages (monocytes) were capable to take up aged PMNs or their enzymes after a maturation step. These recruited macrophages also showed 35 increased fibrinolytic activity and less procoagulant activity than resident macrophages. These changes would maximize fibrin removal (Dal Nogare and Towens, 1990). The fact that fibrin could be easily removed had direct implications in resolving pulmonary inflammation since it permitted more efficient macrophage motility, thus enhancing their cleansing function. Also decreased formation of fibrin deposits, which may act as foci for collagen and fibroblast attachment, reduces the risk of generating lung fibrosis (Dal Nogare and Towens, 1990). Macrophage activation is not always a beneficial response. It was clear that activation may also result in injury to the lung parenchima and induce interstitial fibrosis (Brieland et al., 1987). Secretory products of macrophages, including degradative enzymes such as acid hydrolases and reactive oxygen metabolites were reported to both directly and indirectly initiate acute lung injury (Brieland et al., 1987). In addition, other potential mechanisms of macrophagemediated lung injury include: production of chemotactic and vasoactive complement fractions (Cab), generation of arachidonic acid metabolites, release of plasminogen activator, secretion of cytokines, and release of lysosomal degradative enzymes (i.e., elastase, mucopolysaccharidases, collagenase) (Fantone and Ward, 1984). It was proven that most of the damage detected in lungs during the initial phase of the inflammatory process was due to increased production of Oz by these cells, and this was caused by a direct increase in the membrane-bound oxidase activity in these cells (Brieland et al., 1987). In addition, an increase in the secretion of acid hydrolases (A/-acetyl -p-D glucosaminidase) was observed (Brieland et al., 1987). It was also proven that mononuclear macrophage cells that arrived from the blood exhibited increased generation of oxygen free radicals as well as acid hydrolases, when compared with the activated resident pulmonary macrophages (Brieland et al., 1987). Abnormal macrophage activation was described as the central alteration in the pathogenesis of several diseases, such as sarcoidosis and granulomatous inflammation of the lungs (Pinkston et al., 1983; Robinson and McLemore,1985; Bitterman et al., 1986). Sarcoidosis was described as a disorder characterized by excessive macrophage-mediated helper T cell activation, excessive ylFN synthesis, and secretion of profibrotic and destructive substances by activated macrophages (Pinkston et al., 1983; Robinson and McLemore, 1985; Bitterman et al., 1986). In granulomatous inflammation of the lungs, the presence of highly irritant, indigestible or at least poorly degradable, persistent unanimated or living substances, elicited the accumulation and activation of macrophages (Adams, 1983). Activation of these cells , as it was mentioned above, determined the generation of substances destructive to the lung parenchima, secretion of cytokines, accumulation of T cells, and synthesis of substances (e.g., fibronectin and alveolar macrophage-derived growth factor) that promoted the migration and proliferation of fibroblasts (Adams and Hamilton, 1988). Interaction of pathogenic microorganism with alveolar macrophages Bacteria The effect of bacterial species on the alveolar macrophages are mediated, preferentially through the secretion of toxic products. There are reports that link bacterial endotoxin and dysfunction of alveolar macrophages. For instance, in one experiment, Sprague-Dawley rats were injected intravenously with either placebo or 5 mg/kg of E. coli lipopolysaccharide B. 37 Two hours after treatment, the rats were challenged with S. aureus and bacteriologic examination was performed immediately and at 4 hours after bacterial challenge (Harris et al., 1988). Rats pre-treated with endotoxin showed a significant decrease in pulmonary bactericidal activity when compared to aerosol-challenged control rats, and this was interpreted as a defect in alveolar macrophage function mediated by the application of endotoxin. Increased production of free radical oxygen species In endotoxintreated alveolar macrophages was also shown, when compared with control untreated cells. (Harris et al., 1988). In another experiment, dogs were given 2 or 20 mg/kg of Escherichia coii endotoxin and several cellular functions of alveolar macrophages were compared with those from control dogs (Jacobs et. al.,1986). Adherence of alveolar macrophages from the endotoxin-treated dogs was significantly reduced. In addition, alveolar macrophages from endotoxintreated dogs showed significantly greater production of hydrogen peroxide, greater peaks in chemiluminescence, reduced phagocytosis and a diminished ability to kill cell-associated S. aureus and E. coii overtime (Jacobs et al., 1986). In another experiment it was proven that generation of oxygen radicals, especially hydrogen peroxide (H2O2), inhibited phagocytosis and superoxide anion production by alveolar macrophages in a irreversible and concentrationand exposure time- dependent fashion. These authors suggested that ATP depletion may play an important role in the described H2O2 toxicity for alveolar macrophages (Jacobs et al., 1986). This suggestion was proven later (Oosting et al.,1990). These authors showed that comparable concentrations of H2O2 caused an irreversible depletion of cellular ATP, reduction of phagocytosis, and production of superoxide by alveolar macrophages . In addition, the time 38 course of ATP depletion and induction of impaired alveolar macrophage function were similar to that described by Jacobs et al. (1986). These authors also indicated that in view of the fact that H2O2 may react with a large variety of biological substances, other possible toxic lesions may not be excluded as underlying mechanism for HaOa-induced inhibition of the phagocytic and killing functions of these cells (Oosting et al.,1990). Several respiratory bacterial pathogens caused impairment of alveolar macrophages by resisting killing. These microorganism are able to multiply inside of the macrophages causing the death of the cell. Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. It was shown that under in vitro conditrans, this bacterium was capable of multiplying to 2.5-4.5 logs/ml, and result in the destruction of the monocyte monolayers (Horwitz, 1987). It was reported that L pneumophila inhibited the phagosome-lysosome fusion, thus evading the killing mechanisms of macrophages (Horwitz, 1987). Respiratory bacterial pathogens of cattle such as Pasteurella hemoiytica and Haemophilus somnus exerted their pathogenic effect on alveolar macrophages through the production of species specific cytotoxins (Corbeil and Gogolewski, 1985). The cytotoxin of P. hemoiytica impaired phagocytosis, killed macrophages, was specific for ruminant leukocytes and was more cytotoxic if opsonization took place. In addition,P. hemoiytica also avoided phagocytosis by means of their capsule. In effect, a high molecular weight capsular fraction of this bacterial species has been shown to adversely affect bovine neutrophil phagocytic function (Corbeil and Gogolewski, 1985). 39 Viruses The effect that virus infection has on alveolar macrophage activity was well documented by Bryson et al. (1983). These authors described parainfluenza-3 (PI-3) virus replication in bovine alveolar macrophages under in vivo conditions and together with the information reported by Breeze and Liggitt (1989), a detailed information on the cellular events induced by the viral replication in BAM is now available . For instance, it was shown that upon infection with PI-3, changes occurred in the arachidonate acid (AA) metabolism (Breeze and Liggitt, 1989). These changes included the elevated production of some of the AA derivatives, most notably PGE2 . It was postulated that these changes were due either to increased phospholypase A activity or to interaction of the virus with the membrane of the cell (Breeze and Liggitt, 1989). It was also shown^ that some of these metabolites, especially PGE2, were inhibitory to phagocyte functions (Laegreid et al., 1989). By using primary cultures of BAM infected in vitro with PI-3 virus, these authors showed that PI-3 virus-infected BAM killed significantly fewer Staphylococus epidermidis than uninfected controls (Laegreid et al., 1989). This inhibitory effect was reverted by the addition of cycloxygenase pathway inhibitors (i. e., indomethacine, pyroxicam ) 1 hour prior to the assay. In addition, they showed that phagosome-lysosome fusion was severely impaired in virus-infected BAM and, pre-treatment of virusinfected BAM with indomethacine significantly enhanced the percentage of cells expressing phagosome-lysosome fusion activity (Laegreid et al., 1989). Other researchers have shown that the blockage of b-1 surface receptors caused the inhibition of reactive oxygen species-dependent chemiluminescent reactions in BAM. Infection of BAM by PI3 virus or BHV-1 was shown to cause impairment of b1-receptor function (Ogunbiyi et ai., 1988). Based on these findings, it was postulated that infection by these virus species render the BAM unable to generate oxygen reactive products, inhibiting the oxygen-dependent killing mechanism of these macrophages (Ogunbiyi et al., 1988). Treatment of BAM with levamisole, reverted the blockage of the b-1 receptors, rendering the macrophages as functionally active as the non-infected control cells (Ogunbiyi et al., 1988). Similarly, it was shown that infection of porcine alveolar macrophages with pseudorabies vims (PVR) caused depression in some of the macrophage functions (Iglesias et al., 1989). It was shown that phagocytosis of yeast was significantly reduced in the alveolar macrophage cultures. It was also shown that phagosome-lysosome fusion and O2 release after stimulation with opsonized zymosan was significantly reduced in all the PRV-infected cultures (Iglesias et al., 1989). These authors concluded that the inhibitory effect of PRV infection on alveolar macrophage bactericidal functions may explain the increased susceptibility of PRV-infected pigs to bacterial pneumonias (Iglesias et al., 1989). In another work it was shown that infection of BAM with PI-3 or BHV1 caused a significant reduction in the percentage of Fc and Cab receptors . The alveolar macrophages obtained after virus inoculation were significantly impaired in their ability to phagocytize opsonized S.epidermidis, but were able to kill ingested bacteria (Brown and Ananaba, 1988). In other work, the same group of researchers obtained similar results under in vitro and they reported a significant reduction in the Csb mediated phagocytic index in alveolar macrophages inoculated with BHV-1 or PI-3 (Brown and Shin, 1990). 41 The overall effect of BHV-1 infection on BAM and their function on the immune response in calves was documented by Tsevtkov (1990). Results from his research indicated that the number, viability and differential distribution of the BAM and their phagocytic and microbicidal ability showed a tendency to decrease during the acute phase of infection (2nd-5th days after infection). This was followed by a slow restoration during the period of convalescence (by the 15th day after infection). Some parameters of systemic immunity.(e.g., number of plaque-forming cells, rosette-forming cells, differential and absolute number of leucocytes in the peripheral blood) also showed similar dynamics (Tsevtkov,1990). It was stated that changes in the local and systemic, and in the cell and humoral immunity were connected to the immune suppressive properties of BHV-1 (Tsevtkov, 1990). Perhaps the best example to understand the central role of the alveolar macrophage in lung defense and the effect of some virus infections is given by a disease where the alveolar macrophage's function is missing. Good examples are the respiratory complications in patients with acquired immunodeficiency syndrome (AIDS). In this disease, the frequency of pulmonary infections suggested that pulmonary host defenses were compromised (Beck and Shellito, 1989). The defense capabilities of the upper and lower airways, which prevent the majority of infectious organisms from reaching the lungs in healthy individuals, were diminished in AIDS patients. In the lower respiratory tract, direct infection of alveolar macrophages with HIV altered clearance of microorganisms (Beck and Shellito, 1989). In addition to a direct effect on the ingestion and killing events, it was shown that soluble signals secreted by activated alveolar macrophages required to activate other macrophages and accessory cells were deficient (Cox et al.,1990). Without an adequate amount of these signals, lung defenses cannot be mounted properly. In addition, peripheral T and B lymphocytes from AIDS patients do not respond to antigen normally, and the resulting defects in cellular and humoral immunity may impair defenses against a variety of pulmonary pathogens (Beck and Shellito, 1989). In addition, the defective microbicidal function of PMN's from AIDS patients, and the reduced migration of blood leukocytes into the lungs result in an insufficient cellular response to an infectious challenge (Beck and Shellito,1989). Mycoplasmas One of the most intriguing aspect of the interaction between mycoplasmas and macrophages is their ability to colonize the surface membrane of the macrophages and actively proliferate in this location without being ingested. This phenomenon was initially described by Jones and Hirsch (1971) and Jones et al. (1972) with M. pulmonis, and it was noticed that the mycoplasmas bound to the phagocyte surface proliferated and were not phagocytized unless anti-mycoplasma opsonizing antibodies were added. These authors proposed the presence of an antiphagocytic mycoplasmal surface protein (Jones et al.,1972). Similar observations were reported with M. arthhtidis, but with this mycoplasma species, only partial opsonization was achieved upon the addition of anti-mycoplasma antibodies (Cole and Ward,1973; Davis et al.,1980). It also was reported that incubation of macrophages with M. hominis and M. arthritidis resulted in diminished opsonization of Escherichia coli, a microorganism that was readily ingested by these cells. This was taken as indication of direct impairment of the phagocytic functions of these cells by the mycoplasma species used in this experiment (Cole et al., 1985). Suppression of lymphoid cell function has been reported in several mycoplasma species. The first report on this effect was described with M. pulmonis. It was shown that the addition of this mycoplasma to human peripheral blood treated with phytohemagglutinin (PHA) resulted in inhibition of the mitogenic effect detected otherwise (Copperman and Morton, 1966). This effect was reversed upon removal of the mycoplasma and supplementation with fresh medium and PHA. It was demonstrated that this effect was due to depletion of arginine by mycoplasmas from the growth medium, rather than a direct effect of the microorganism of lymphocyte functions (Barile and Levinthal, 1968). The same authors documented the inhibition of PHA stimulation on lymphocytes by non-fermentative mycoplasma species (e.g., M orale and M. arthritidis), and these results were confirmed by Morton et al. (1968) and Simberkoff et al. (1969), who reported that the inhibitory effect was promoted by extracts obtained from non-fermentative species of mycoplasma. It was also shown that the inhibitory moiety was heat-labile and papain sensitive, suggesting a proteinaceous composition (Simberkoff et al., 1969). Mycoplasma species involved in enzootic pneumonia in calves, (i.e., M. dispar and M. bovis) have been reported to be immunosuppressive. Such property has been supported by several experimental and in vivo observations (Bennet and Jasper, 1977; Howard et al., 1987). Among these, it was reported that subcutaneous inoculation of killed M. dispar Induced a poor immune response in calves compared to that obtained with M. bovis (Howard and Gourlay, 1983). In addition, the Inoculation of killed preparations of M. bovis and M. dispar resulted in a reduced antibody response to M. bovis, lower than that observed when M. bovis was inoculated alone (Howard et al., 1987). In another study, mycoplasma species pathogenic for the respiratory tract of cattle {M. dispar, M. bovis ), and a non-pathogenic respiratory mycoplasma species {M. bovirhinis ), were studied for their effect on the response of lymphocytes to mitogens (Finch and Howard, 1990). It was found that live M. dispar and M. bovis had inhibitory effects on the stimulation of these cells by PHA. When killed M. dispar was used, little effect was noticed, which was in contrast with the elevated inhibitory effect shown with killed M. bovis. Neither live nor killed M. bovirhinis had a demonstrable effects on lymphocyte responses to PHA. In experiments with live M. dispar and M. bovis it was noticed that there was no alteration of the lymphocyte viability, and there was no evidence of mycoplasma replication. Live M. dispar also inhibited the mixed leucocyte response (MLR) (Finch and Howard, 1990). Similarly, live M. bovis also showed the same effect on MLR, but in contrast with the previous experiment, heat- killed and formalin-killed M. dispar and formalin-killed M. bovis were also inhibitory. As before, there were no alterations in leucocyte viability and no indication of mycoplasma replication in these experiments containing viable mycoplasma (Finch and Howard,1990). In another set of experiments, the effect of filtered supernatants from bovine peripheral mononuclear cells inoculated with M. dispar and M. bovis was tested. It was observed that supernatants from cultures containing viable mycoplasma inhibited the MLR. The same effect was observed with formalin killed but, in contrast, no effect on MLR was observed whit supernatant from mononuclear cells with heat killed M. bovis. Supernatants from mononuclear cells treated with heat or formalin killed M. dispar d\d not show any suppressive effect on MLR (Finch and Howard, 1990). The results obtained from these experiments indicated that viable M. dispar or M. bows was required for the inhibition of MLR and the molecule(s) responsible for such effect were heat sensitive in both mycoplasma species (Finch and Howard, 1990). The correlation between mitogenicity, induction of peribronchial cuffing, and lymphocyte accumulation was previously shown with M. pulmonis (Cole et al., 1985). The results reported by Finch and Howard (1990) did not agree with this observation, since peribronchial cuffing and lymphocyte accumulations were described in M. bovis pneumonias, yet this mycoplasma had inhibitory effects on lymphocyte proliferation. These authors proposed that these pathologic changes could by induced by induction of TNF and interleukins, which in turn, caused necrosis and accumulation of leucocytes around the airways (Finch and Howard,1990). While the induction of TNF by macrophages and reproduction of the pathological changes seen in the natural disease was proven with TNF or a carbohydrate moiety produced by Mycoplasma mycoides subsp. mycoides (LC) (Rosendal, 1990), it is not known if macrophages infected with M. bovis can be induced to produceTNF. The induction of a inhibitory state by the supernatants of cultures with viable mycoplasma (M dispar or M. bovis) without changes in the viability of leucocytes was taken as evidence of the presence of a heat-labile factor(s) produced by the mycoplasma that modified the cellular function of the cells (Finch and Howard,1990). Similar observations were made with M hyopneumonia, where azide-treated mycoplasma were found to suppress the response of porcine lymphocytes to mitogens, an effect that was also heat-labile (Kishima and Ross, 1985). 46 Various mycoplasma species have been shown to possess T cell and B cell mitogenic activity (Cole et al., 1981; Cole et al., 1985; Naot et al., 1987). It also was reported that mycoplasma induced the cytotoxic activity of NK cells, cytotoxic T-cells and macrophages (Wayner et al., 1984; Lowenstein and Gallily, 1984). Recent reports indicated that several mycoplasmas may induce production of tumor necrosis factor by macrophages. Such stimulation was mediated in a Ca2(+)-dependent but not protein kinase C-dependent pathway and the induction of this cytokine occurs at the transcription level (Gallily et al., 1989; Sugama et al., 1990). It was also reported that some mycoplasma species can induce expression of MHC class I and II antigens, alter the production of IIL-1, induce production of IL-2, and induce the production of granulocyte-macrophage colony-stimulating factor from human lymphocytes (GM-CSF). It was shown that purified membranes of M. arginini and M. arthritidis but not M. pulmonis induced the proliferation of macrophages throughout the production of GM-CSF by lymphocytes (Cole et al., 1990). In this study, it was shown that the factor that mediates this effect was a mycoplasmal membrane protein of approximately 60 kDa in size. The significance of these host responses to mycoplasma products was documented by the involvement of mycoplasmas in several animal diseases, including atypical pneumonia and arthritis. It was postulated that the mechanisms for the pathogenic effects of mycoplasmas included the activation of host immune cells. The fact that M. arthritidis produces a superantigen that induces autoimmune reactions through the activation of T cell clones, suggests the direct participation of mycoplasma in the pathogenesis of these autoimmune conditions (Cole et al., 1990). 47 It is not clear at this point whether the activation of the host immune cells by mycoplasma products leads to development of autoimmunity or if they simply represent an exacerbated inflammatory response. I 48 PART i. Impaired Function of the Mucociliary Apparatus of Calves Infected with Mycoplasma dispar ï 49 Impaired Function of the Mucociliary Apparatus of Calves Infected with Mycoplasma dispar Raul A. Almeida, D.V. M., M. S. Ricardo F. Rosenbusch, D. V. M. , Ph. D. From the Veterinary Medical Research Institute, and the Department of Microbiology, Immunology and Preventive Medicine, Iowa State University, Ames, Iowa, 50011. Supported in part by Iowa Livestock Health Advisory Council, grant No.109-05-17 50 SUMMARY Infection of tracheal organ cultures with Mycoplasma dispar results in degeneration of respiratory epithelial cells with loss of ciliary activity. To assess the effect that these changes have on the clearance of bacteria from the respiratory tract, the tracheobronchial clearance of a suspension of Serratia marcescens was determined in calves before and after infection with M. dispar. Tracheobronchial swab samples were obtained at various times after deposition of the marker bacteria. The clearance was significant by 3 hours after inoculation and only few colonies were detected 4 hours after challenge with the marker bacteria. In contrast, in the same animals, 5 days after intratracheal exposure with M. dispar, clearance of the marker was impaired beyond 4 hours after inoculation. Histopathological and scanning electron microscopy (SEM) studies of infected lungs revealed that as a result of infection with M. dispar, areas of degeneration and destruction of the respiratory ciliated epithelium occur in intermediate and small airways. These lesions may be responsible for the altered clearance observed in these mycoplasma-exposed calves. 51 INTRODUCTION The sterility of the internal portions of the respiratory tract is achieved and maintained by the synergistic interaction of numerous defense mechanisms. The mucociliary apparatus is one among these, and in this regard, its function is to prevent the contact of harmful substances and infectious agents with the epithelial surfaces of the airways by removing these harmful agents from the respiratory tract. These effects are achieved by the interaction of the trapping capacity of the mucous layer and the propelling effect of the ciliary blanket. The initial steps in the pathogenesis of pneumonic processes are the multiplication of pathogenic microorganisms in the upper regions of the respiratory system, which is followed by the overcoming of the mucociliary apparatus and dissemination into deeper regions of the lungs. Pathogenic bacteria have developed means to circumvent this first defense barrier by reducing the viscoelastic properties of the mucous layer and by impairing ciliary activity (6, 7). Some bacterial species such Pasteurella multocida and Actinomyces pyogenes break down the mucus layer through the production of neuraminidase or extracellular proteases, thus rendering the mucus less viscous and ineffective for the trapping and removal these or other infectious agents (4, 5). Other respiratory pathogens, such as mycoplasmas, may cause ciliostatic and ciliotoxic alterations which impair the mucociliary clearance (6, 7). Using a bovine tracheal organ culture system it was shown that Mycoplasma dispar, a bovine respiratory pathogen, caused alterations of the ciliary apparatus which include degeneration of the respiratory epithelial cells and loss of the ciliary activity (9, 10). Since this ciliostatic action of M. of/spar may be 52 an important predisposing factor in the developing of lung infection in cattle, in the present report we extend these observations to in vivo conditions by investigating the tracheobronchial clearance of a marker bacteria in calves experimentally inoculated with M. dispar. i' 53 METHODS Animals Ten calves of 2 to 2.5 months of age were procured from a herd negative to mycoplasmas by serology and by culture of nasal swabs. Calves were housed in individual isolation rooms and daily inspected during 7 days. Inspection consisted in verification of the general condition of the calves, presence of signs of respiratory infections (increased respiratory rates, nasal discharge, cough), presence of gastrointestinal alterations (feces consistency), and apetite. Microorganisms. Mycoplasma dispar SD-0 (11) at passage level 7 was used in this experiment. Mycoplasmas were grown in modified Friis broth medium (12), washed three times with PBS, resuspended at 100 X concentration in 1 ml aliquots of Friis broth medium, frozen and kept at -70 C until use. To challenge calves, aliquots of the mycoplasma suspension were resuspended in sterile PBS to a titer of 10^° color changing units/ml (ccu/ml). Serratia marcescens strain NIMA used in this experiment was grown as was described by Veit et al., 1978 (13). Briefly, sterile tryptose broth was inoculated withS. marcescens and incubated at room temperature for 96 h. Aliquots of the broth were used to inoculate tryptose agar plates which were incubated for 36 h at room temperature. One single red colony was picked from a selected agar plate and used to inoculate 100 ml of tryptose broth. After 24 h of incubation at 37 C, cultures were centrifuged at 5000 X g for 20 min, resuspended in 20 ml of lactose-glutamate diluent (14), and 1 ml aliquots of the broth were quick-frozen and kept at •70°C until use. 54 a To challenge calves, one aliquot of the suspension was thawed at 37°C and resuspended in PBS (pH 7.2) to an OD of 1.045 at 600 nm (Spectronic, Milton Roy, Rochester, NY). Experimental Design. Calves were assigned at random to each of 3 groups. Group A consisted of calves that were inoculated transtracheally (between the upper and mid-third of the tracheal length) with 10 ml of a suspension of S. marcescens. These calves were put under sedation with 0.5 mg/lb Xylazine given I.M. (Mobay Corp. Shawnee, KA). Mucosal swab and tracheal samples were obtained with 27 inch-long occluded swabs (Accu-Med Corporation, Pleasantville, NY) with the aid of a laryngoscope. Briefly, while holding the head and the neck of the calf in a horizontal position, the swab was carefully introduced through the larynx into the trachea. Once the occluded end of the swab contacted the tracheal bifurcation, it was retracted 2-3 cm, the tip exposed and kept in that position for 15 to 20 seconds. Rotational movement of the swab was avoided to reduce chances of producing physical trauma to the tracheal mucosa. Four samples were obtained at one hour intervals, and after the last sampling time, calves were inoculated transtracheally with 10 ml of a M d/spar suspension in PBS (lO'io ccu/ml). After 5 days, calves were challenged again with S. marcescens following the routine described previously. Tracheal swab samples were placed in dry tubes and kept on ice until processing, which occurred 5 to 10 min after obtain the sample. Calves were euthanized 24 hours after the last sampling. Group B consisted of 2 calves that were subjected to a routine similar to that described above, but in this case no experimental infection with M dispar 54 b was performed. Calves were euthanized 24 hours after the last sampling. Group C consisted of 2 calves that were inoculated transtracheally with 10 ml of a M. d/spar suspension in PBS (1010 ccu/ml). These calves were euthanized 5 days after infection. Determination of the tracheobronchial clearance of S. marcescens. To enumerate the number of S. marcescens, swabs taken from calves of group A and B were placed in microtubes with 1 ml of diluent solution (0.5 M sucrose, 50 mM phosphate buffer pH 7.0, and 50 mM MgS04 ), briefly centrifuged (1 min at 14,000 X g) and 10 -, 100 -, 1000 - fold dilutions were performed in the same diluent. An additional dilution ( 10000-fold) was added to samples obtained from M. d/spar infected calves. One hundred microliters of undiluted samples and of .each dilution were plated by triplicate on tryptose agar plates using standard spread plate methodology and incubated at room temperature for 48 hours. Pigmented colonies were counted and the logarithm of the number of colonies/swab was calculated. Comparisons were made between the data obtained before and after infection with M. dispar. Necropsy Procedures. All the calves were euthanized by IV administration of an overdose of sodium pentobarbital (Fort Dodge Laboratories Inc., Fort Dodge, la). Lungs were aseptically removed from the thoracic cavity and after recording the percentage of lung consolidation, samples from several predetermined areas were processed for histopathology, SEM, and mycoplasma and bacterial isolation. Samples were placed in 10% buffered-formalin or vigorously washed with 55 0.075 M Sorensen's phosphate buffer (pH 7.2) to remove mucus and placed in ice-cold and freshly prepared 3%glutaraldehyde solution for histopathology and SEM, respectively. Samples for mycoplasma and bacterial isolation were placed in sterile containers and kept on ice until processing. Histopathological Procedures. Formalin fixed lung tissue specimens were dehydrated, embedded in paraffin, sectioned at 5 ^m, mounted on glass slides and stained with hematoxilyn-eosin. SEM procedures. Tissues fixed overnight in 3% glutaraidheyde were sequentially treated with two changes of osmium tetroxide and 5% tannic acid solution, using the method described by Sweney and Shapiro (8). Tissues were dehydrated in ethanol, critical point dried from absolute alcohol, and sputter-coated with goldpalladium. Microbiology Procedures. Lung tissues were suspended in PBS (pH 7.2) and triturated in a tissue grinder. The homogenates were centrifuged at 100 x g for 10 min and ten fold dilutions of the supernatants were inoculated in Hayflick broth (pH 6.8) and Modified Friis broth tubes (12). In addition, 0.2 ml of undiluted lung homogenate was placed in Friis and Hayflick agar plates and spread with the aid of a Drigalsky spatula. Tubes and agar plates were placed at 37' C in a CO2 incubator and checked for mycoplasma growth during 10 days. Suspect broth cultures were transferred to the corresponding agar plates and colonies of mycoplasmas were identified by 56 the IFA technique. For bacterial isolation, 7% bovine blood agar plates and tryptose agar plates were inoculated with 0.2 ml of lung homogenates and incubated at 37 C for 48 hours. Bacterial isolates were identified using a Minitek identification system (BBL, Cockeysville, IVID). Statistical Analysis Data are reported as mean ± standard deviation. Data were analyzed by paired f tests. Significance was accepted for a comparison when the p value was less than 0.05. 57 RESULTS Clearance of Serratia marcescens Recoveries of S. marcescens at different times before and after the infection with M disparare shown in Figure 1A and 1 B. The tracheobronchial elimination of S. marcescens was significantly slower after infection with M. d/spar compared with that observed in animals not infected with mycoplasma. Before infection with M. dispar, rapid elimination of the bacterial marker was detected 3 hours after inoculation and at 4 hours, only few colony forming units were detected. In contrast, in M. dispar infected calves, recovery of the marker was high at 3 hours and unchanged 4 h after inoculation with S. marcescens. The pattern of elimination observed after M of/spar infection, suggested that clearance of the marker bacteria would extend well beyond 4 hours after inoculation. Differences in recoveries of S. marcescens before and after infection with M. of/spar were significant (p< 0.05). In addition to these changes, increased bacterial counts were observed overall on M. dispar infected calves when compared to those obtained in the non-mycoplasma infected status. The pattern of tracheobronchial elimination of the bacterial marker in calves given saline instead of mycoplasma (group B) was similar after the first and second inoculation (Fig. 1 B), and was similar to that detected in principal calves before infection with M. dispar. Clinical Signs On day 2 after inoculation with M. d/sparcalves were dull and depressed. These signs remained for 24 hours affecting 6 calves (5 calves of group A and Figure 1. Means of the number of CPU of S, marcescens recovered from the principal and control calves at each sampling time. A) Lower line (open squares) corresponds to determinations made before infection with Mycoplasma dispar and upper line (close squares) correspond to those made after infection with Mycoplasma dispar. B) Colony forming units of S. marcescens detected on the control calves at each sampling time. Open circles correspond to determinations made after the first challenge and closed circles line correspond to those made after the second challenge with the bacterial marker. 59 B) Recoveries in caives from group B 81 A)Recoveries in caives from group A 7- 6- I « 4 3- T—'—I —r 2 3 Time (hours) 4 -I r—' 1 1 2 1 3 ' Time (hours) 1 4 ' 1 5 60 one of group C) of the 8 calves inoculated with M. dispar. No other clinical signs of disease were observed. Gross Post-Mortem Findings At necropsy, macroscopic lesions were confined to the lungs. The lungs of the calves infected with M. d/spar contained consolidated areas which occupied up to 1-2% of the lung parenchyma (Table 1). These areas of consolidation were located mainly in the cranial lobes and were dark red. No exudate was expressed when these areas were pressed. Table 1- Histopathological and Microbiological Findings in Experimental Calves. Calf Qoup Pathology Consolidation Microbiology BronchiolUs Emphysara M dispar Smanxsoem f/o) 1 a 1 + + + + 2 a 2 + + + + 3 a 0.5 + - + + 4 a 1 + - + + 5 a 1 + + + + 6 a 1 + - + - 7 b 0 - - - - 8 b 0 - - - - 9 c 1 + + + - 10 c 2 + + + - 61 Histopathological and SEM findings Histopathological studies performed in calves infected with M. of/spar showed that early changes were detected mainly in the respiratory epithelium of medium and small bronchioles. These alterations included degeneration of ciliated cells with loss of cilia, pyknosis and cytoplasmic shrinkage. The lumen of bronchioles and smaller airways from M. cA'spar infected calves contained a considerable amount of cellular debris and polymorphonuclear cells (Fig. 2). Also, a moderate infiltrate of inflammatory cells, mainly PMN's and macrophages, was noticed in the submucosa of these airways. In contrast, ciliated epithelium from large airways was not altered. No lesions were detected in calves of group B (Fig. 3). Scanning electron microscopy of the airways of calves infected with M. dispar showed ciliary clumping with presence of inflammatory cells (Fig. 4). Also patchy areas with loss of the ciliated epithelium and presence of inflamatory cells were seen in these airways (Fig. 5). Extruded ciliated cells were observed in the lumen of the airways. No alterations were detected in the epithelium of large airways (Fig. 6). No lesions were observed in animals challenged with S. marcescens alone (data not shown). Microbiology Findings. Mycoplasma dispar was isolated from the lungs of all the calves of group A and C. In addition, S. marcescens was isolated from the lungs of 5 calves belonging to group A. No other bacteria was isolated from any of the lungs of the calves in these experiments. No mycoplasmas were isolated from the respiratory tract of calves belonging to group B (Table 1 ). Figure 2. Small bronchiole from a M. d/spar infected calf showing alteration of the ciliated eipthelium and presence of polymorphonuclear cells in the lumen. (H&E, 450 X). Figure 3. Small bronchiole from a calf of group B without degenerative changes. (H&E, 450X). 63 Fîg. 4. Scanning electron micrograph of small bronchiole of calves infected with M. d/spar showing clumping of the ciliary blanket with inflammatory cells on surface (SEM, 940 X). Figure 5. Scanning electron micrograph of a small bronchus of a calf infected with M cf/spar showing areas with loss of the ciliated epithelium and presence of inflamatory cells. (SEM, 600 X). Figure 6. Scanning electron micrograph of a large bronchus of a calf Infected with M. d/spar showing no apparent damage of the ciliated epithelium (SEM, 3600 X), 67 I 68 DISCUSSION The study of lung clearance has been used to delineate factors and conditions that determine the retention of organic or inorganic particulate markers in the respiratory tract. In some of these studies, clearance of the particulate marker has been studied using the technique of sequential slaughter (13,15,16, 17,18,19,20). This method has a major disadvantage, which Is the slaughter of a large number of animals and the obtaining of multiple samples from each individual, adding more cost and source of variation to the system. Using a different approach, mechanical clearance has been assessed by external monitoring of inhaled, inert radioactive aerosols (7,21,22,23). A variation of this technique was described by Viso et al. (24), In which the clearance of a opportunistic bacterial species of low pathogenic potential was sequentially determined in the same animal during a period of time. In the present investigation, a modification of this method was used to determine the effect of M d/spar infection on clearance of S. marcescens and it could be shown that animals infected with this mycoplasma species presented reduced clearance of the bacterial marker. Four hours after Inoculation of M. dispar-Uee calves, only a few colonies of S. marcescens were recovered. In contrast, in the same calves and after infection with M. dispar, the tracheobronchial elimination of S. marcescens was prolonged beyond 4 hours after inoculation of the bacterial marker. In addition, the isolation of S. marcescens was possible from the lungs of calves from group A but not from group B, which was indicative of the impaired tracheobronchial clearance and increased retention of the bacterial marker in calves infected with M. dispar. L 69 It was previously shown that exposure to virus (25) or to mycoplasmal agents (20) will retard the clearance of inhaled bacteria. In these studies the impairment of the clearance was presumed to be due to lesions of the mucociliary apparatus caused by these pathogens. The results presented here are comparable with these previous reports, in that the infection with M. dispar alters the normal tracheobronchial clearance in calves. Similarly, it is possible that the decreased clearance could be the result of the lesions that M. dispar produced to ciliated epithelium of the respiratory tract. In previous studies, conventionally raised and gnotobiotic calves were experimentally infected with M dispar and killed between 3 to 5 weeks later. Histophatological results from these studies included the presence of exudative bronchitis and interstitial alveolitis centered around the bronchioles with moderate to negligible production of peribronchial cuffs (3, 2 ). The histopathological descriptions in calves naturally infected with M. dispar included bronchitis and bronchiolitis obliterans with large cuffs of mononuclear cells around these ainways. Mycoplasma of/spar colonies were detected in the surface of the affected airways (11). Changes in the ultrastructure of the bronchial epithelial cells included loss of the cilia and cytoplasmic protrusion into the airway lumen (1 ). Lesions described from in vitro studies, in which expiants of bovine tracheas were inoculated with M. dispar, included profound damage to the ciliary epithelium with pyknosis, sloughing and flattening of the cells with consequent loss of the ciliary activity (9). Based on these results, it was proposed that M. d/spar colonizes the bronchioles in close relationship with the cilia and microvilli of the epithelial cells, altering their metabolism and stopping the ciliary movements with necrosis and desquamation of the cells (9). Results from histopathological and SEM studies of the lungs of calves Infected with M. dispar alone or in combination with S. marcescens as presented here, indicated destruction of respiratory ciliated surfaces and degeneration of the respiratory ciliated epithelial cells mainly in small and medium size airways. Minimal alveolar reaction surrounded these damaged ainways and no cuffing reaction was seen. The slow development of the pulmonary lesion which correlated with the slow growth of M. dispar was previously described (3), and these can be taken here to explain the mild character of the alveolar lesions described. Previous descriptions of lung damage caused by M. dispar have focused on late stages of lesion development, in contrast these experiments provide a description of the lesions that develop in the early stages of the infection. Based in the results of the present communication, it is likely that these circumscript lesions affecting the ciliary blanket are related to the strong affinity shown by this mycoplasma species with the surface structures of the respiratory mucosa (9). These lesions were localized mainly in medium and small airways and not in bronchii or the trachea, contrasting with the lesions associated with M. hyopneumoniae infections which were detected in epithelial ciliated cells of trachea and in main bronchii with minimal damage to the bronchioles or medium size airways epithelium (27). The decreased clearance of S. marcescens can also be partially explained by changes in the composition of the mucous layer. It was reported that the increased numbers of goblet cells detected in the respiratory tract of pneumonic calves resulted in decreased viscosity of the mucous layer and reduced the efficinecy of trapping and removing inhaled microorganism or particles out of the respiratory tree (1). This effect which was considered non-specific for mycoplasmal pneumonias, may act in conjunction with the destruction of ciliary cells. An interesting result from the present study was the finding that damage of the ciliated epithelium was more evident in small bronchioles than in bronchi or main ainways. This feature can be explained by the slower mucus transport and the paucity of ciliated cells present in small and medium airways, which may allow more close contact between the mycoplasma and ciliated structures. The retarded mucus flow caused by damage of the ciliary blanket not only will interfere with the removal of inhaled particles but also will not provide the adequate mucus flow to assure the clearance and preservation of the underlaying respiratory mucosa above the damaged segment (21). The final outcome of this process will be the entire reduction of the mucociliary transport in the distal segments of the airways allowing the colonization of M. disparas well as the local proliferation of secondary bacterial invaders. The importance of this latter observation needs to be considered in reference with fast growing, highly pathogenic bacterial species for cattle, such as Pasteurella hemolytica. i 72 The results presented here and the effect that M. of/spar has on the alveolar macrophage phagocytic functions (28), are grounds to reconsider the importance of this agent as a causal and predisposing agent of pneumonic infection in cattle. 73 REFERENCES 1. Allan EM. Some features of pulmonary mycoplasmosis in groups of naturally infected calves. In pages 305-316. Respiratory disease of cattle. A seminar of the EEC programme of coordination of research on beef production held at the Netherlands. Martinus Nijoff, The Netherlands 1978. 2. Friis RN. Mycoplasma disparas a causative agent in pneumonia of calves. Acta Vet Scan 1980; 21:34-42. 3. Gourlay RN, Howard CJ, Thomas LH, and Wild SG. Pathogenicity of some Mycoplasmas and Acholepasma species in the lungs of gnotobiotic calves. Res Vet Sci 1979; 27:233-237. 4. Mueller HE, Krasemann C. Virulence of Pasteurella multocida strains and their neuraminidase production. Zentralb Bakteriol 1974; 299A:391-400 5. Lipsky BA, Goderbreg AC, Tompkins LS. Infections causes by nondiphteric Corynebacteria. Rev Infec Dis 1982; 4:1220-1235. 6. Konietzko N. Mucociliary transport and bronchial inflammation. Z Gesamt Med 1988; 43:323-327. 7. Jarstrand C, Camner P, Philipson K. Mycoplasma pneumoniae and tracheobronchial clearance. Am Rev Resp Dis 1974; 110:415-419. 8. Sweney LR, Shapiro BL. Rapid preparation of uncoated biological specinnens for scanning electron microscopy. Stain Tech 1977; 52:221-227 9. Thomas LH, Howard CJ, Parsons KR, Anger HS. Growth of Mycoplasma bovis in organ cultures of bovine foetal trachea and comparison with Mycoplasma dispar. Vet Microbiol 1987; 13:189-200. 10. Howard CJ, Thomas LH. Inhibition by Mycoplasma dispar oi ciliary activity in tracheal organ cultures. Infect Immun 1974; 10:405-408. 11. Tinant MK, Bergeland ME, Knudtson, WU. Calf pneumonia associated with Mycoplasma d/spar infection. J Amer Vet Med Assoc 1979; 175:812-814 12. Knudtson WU, Reed DE, Daniels G. Identification of mycoplasmatales in pneumonic calf lungs. Vet Microbiol 1986; 11:79-91 13. Veit HP, Farrell RL, Troutt HF. Pulmonary clearance of Serrafta marcescens in calves. Am J Vet Res 1978; 10:1646-1650 14. Rosenbusch RF. Influence of mycoplasma infection on the expression of Moraxella bovis pathogenicity. Am J Vet Res 1983: 9:1621-1624. 15. Lillie LE, Thompson RG. The pulmonary clearance of bacteria by calves and mice. Can J Comp Med 1972; 36:129-137. 75 16. Sebunya TNK, Saunders JR. Pulmonary clearance of Haemophilus pleuropneumoniae and Serratia marcescens in mice. Am J Vet Res 1982; 43:1799-1801. 17. Saunders JR, Sebunya TNK, Osborn AD. Pulmonary clearance of Bacillus subtilis spores in pigs. Can J Comp Med 1983; 47:43-47. 18. Veit HP, Farrell R. Increased pulmonary clearance of Serratia marcescens In calves given intravenous Freund's complete adjuvant. Cornell Vet 1984; 74:269-281 19. Rodriguez LM, Lopez A, Merino-Moncada M, Martinez-Burnes J, Mondragon I. Tracheal versus pulmonary deposition and clearance of inhaled Pasteurella hemolytica or Staphylococcus aureus in mice. Can J Comp Med 1985; 49:323-326. 20. Slauson DO, Lay JC, Castleman WL, Neilsen NR. Acute inflammatory lung injury retards pulmonary particle clearance. Inflammation 1989; 13:185-199. 21. Foster WM, Langenback E, Bergofsky, EH. Measurement of tracheal and bronchial mucus velocities in man: relation to lung clearance. J Appl Physiol 1980; 48:965-971. 76 22. Jones CDR, Bull JR. Deposition and clearance of radiolabelled monodisperse polystyrene spheres in the calf lung. Res Vet Sci 1986; .42:8291. 23. Davies CP, Webster AJF. Deposition and clearance of monodisperse aerosols in the calf lung: Effect of particle size and mucolytic agent (Bromhexine). Can J Vet Res 1987; 51:306-311. 24. Viso M., Espinasse J, Lambert P. Measure de la clairance tracheonbronchiale des bovins a l'aide de 1' aspiration transtracheale. Proceedings of the Fourteenth World Congress on Diseases of Cattle. Dublin, Ireland. World Association for Buiatrics. 1986; 1:509-511. 25. Sellers TF, Schuman J, Boubier C, McCune R, Kilbourne ED. The influence of influenza virus infection on exogenous staphylococcal infection and endogenous murine bacterial infection of the bronchopulmonary tissues. J Exp Med 1961; 114:237-256. 26. Stalheim OHV. Mycoplasmal respiratory disease in ruminants: A review and update. J Amer Vet Med Assoc 1983; 182: 403-406. 27. Mebus CA, Underdahl NR. Scanning electron microscopy of trachea and bronchi from gnotobiotic pigs inoculated with Mycoplasma hyopneumoniae. Am J Vet Res 1977; 38:1249-1254 77 28. Almeida RA, Rosenbusch RF. Interaction between capsulated Mycoplasma dispar and bovine alveolar macrophages. Proceedings of the 91st General Meeting of the American Society for Microbiology. 1991 ; Abst.G-40 pp 140. Dallas, Texas. 78 PART II. Interaction of Mycoplasma disparwWh bovine alveolar macrophages 79 Interaction of Mycoplasma disparwWh bovine alveolar macrophages Raul. A. Almeida, D. V. M., M. S. Ricardo. F. Rosenbusch, D. V. M., Ph. D. Michael. J. Wannemuehler, Ph. D. From the Veterinary Medical Research Institute and the Department of Microbiology, Immunology, and Preventive Medicine, Iowa State University, Ames, Iowa, 50011. Supported in part by Iowa Livestock Health Advisory Council, grant No.109-05-17 r 80 ABSTRACT The capacity to avoid phagocytosis, the impairment of the bactericidal activity of bovine alveolar macrophages (BAM) and the activation of BAM by encapsulated Mycoplasma dispar or purified M. d/spar capsule was investigated. Encapsulated and unencapsulated M. dispar were cocultured with BAM in the presence or absence of antisera prepared against unencapsulated M. dispar or purified capsule antiserum. Unopsonized mycoplasma resisted phagocytosis while only anticapsular antibodies enhanced the phagocytosis of encapsulated mycoplasmas. Bovine alveolar macrophages, cultured in presence of viable unencapsulated or encapsulated M. dispar or purified capsule, were tested for their killing activity on Serratia marcescens and Staphylococcus aureus. Killing of both bacterial species was reduced when BAM were treated with purified capsule or encapsulated M. dispar. In contrast, no reduction in the bactericidal activity was observed when unencapsulated mycoplasma were used. Bovine alveolar macrophages were cultured in presence of purified M. dispar capsule or either live or heat killed encapsulated or unencapsulated M. dispar. The supernatant of these cultures were assayed for tumor necrosis factor (TNF), interleukin 1 (iL-1), and glucose consumption, as indicators of macrophage activation. Tumor necrosis factor and IL-1 were produced by BAM when stimulated with unencapsulated M. dispar but not when encapsulated M dispar or its purified capsule was used. Similarly, glucose consumption was increased in presence of unencapsulated M dispar, but not when BAM were cocultured with encapsulated M. dispar or purified capsule. These results indicate that encapsulated M. dispar or purified capsule exert an inhibitory effect on the activity of BAM. 81 INTRODUCTION Alveolar macrophages are central in the protection of the lower portions of the lungs, operating in many nonspecific modes of defense and augmented by specific immunologic mechanisms. Previous studies have shown that alveolar macrophages ingest, inactivate, and degrade inhaled microorganisms within eight hours of their entrance into alveolar regions (12 ). Such antimicrobial ability of macrophages is centered on their phagocytic capabilities and in the production of potent microbicidal and degradative substances within the macrophage. It has been shown that macrophages have innate functional capacities that extend far beyond phagocytosis. They can act as secretory and regulatory cells, can initiate and prolong inflammatory processes, and can stimulate synthesis of extracellular matrix proteins (4, 14). Thus, they can adjust their response to their microenvironment and exert regulatory activities on other cells such as neutrophils and lymphocytes mounting the defense system that protects the respiratory parenchyma from microbial invasion (4). However, if macrophage function is impaired due to pollutant or microbial exposure, the host-parasite balance is upset and disease ensues. In the absence of specific antibodies, several mycoplasma species can colonize the surface of macrophages without stimulating phagocytosis (2, 5, 8, 18). In contrast, other mycoplasma species were capable of stimulating macrophage activity, induce secretion of cytokines, expression of MHC molecules and release of hydrolytic enzymes from these cells (7, 27). Experiments with M. d/spar have shown that this mycoplasma species can induce immunosuppression when injected in combination with other antigens, and it was postulated that the production of soluble inhibitory factor(s) were responsible for the observed immunosuppression (11). The present studies examined the interaction of M. d/spar with bovine alveolar macrophages and found that M. d/spar capsule exerts a suppressive effect. The bactericidal activity of macrophages as well as the secretory functions of these phagocytic cells were reduced by the presence of encapsulated M. dispar or polysaccharide capsule. MATERIALS AND METHODS Macrophages. Bovine alveolar macrophages were obtained by bronchoalveolar lavage of whole lungs. Briefly, 75-90 day-old calves from a mycoplasma-free herd were sacrificed by intravenous injection of a overdose of sodium pentobarbital (Fort Dodge Inc., Fort Dodge, la.), and trachea and lungs were aseptically recovered for lavage. Two consecutive bronchoalveolar lavages were performed with 400 ml of ice-cold modified Hank's solution (EHBSS: 2.5 mM EDTA, 0.4 gm/l KCI, 0.06 gm/l KH2PO4. 8.0 gm/l NaCI, 0.35 gm/l NaHCOs, 0.09 gm/l Na2HP04.7H20, penicillin G 62 p.g/m, kanamycin 100 |ig/ml,streptomycin 100 p.g/ml, and amphotericin B 50 p.g/ml). The first 10 ml of bronchoalveolar lavage fluid were discarded and the rest of the fluid was gently transferred to a sterile container and kept at 4°C. The suspension was then centrifuged at 200 X p for 10 min at 4°C and the pellet was gently resuspended in Eagles's minimal essential medium (MEM) supplemented with 5% FBS (GIBCO Laboratories, Grand Island, N.Y.), and centrifuged at 200 X gf for 10 min at 4°C. The cell population was depleted of erythrocytes by isotonic lysis with a 0.83% Tris ammonium chloride solution for 5 min at 37°C. Cells were centrifuged as before and the pellet was resuspended in E-HBSS without amphothericin B. The proportion of macrophages was estimated by phagocytosis of latex beads (Poly Sciences, Inc., Warrington, PA), and viability of the cells was checked by Trypan-Blue exclusion. The proportion of macrophages was enriched using the selective adherence of macrophages to plastic surfaces, following the protocol described by Pennline (21). Briefly, cells were spun down (200 X g for 10 min at 4°C) and resuspended in MEM containing 15 % FBS with 62 |ig/ml of penicillin G, [' 84a 100 ng/ml of kanamycin, and 100 ^ig/ml streptomycin, and placed in Petri dishes at a concentration of 3 X 10^ cells/cmZ. The cells were incubated overnight at a 37°C in a 4 % CO2 atmosphere. Adherent cells were then harvested as follows: after washing the monolayer to remove non adherent cells, ice cold E-HBSS was added and allowed to stand at 4°C for 30 min. Cells were then washed twice with warm E-HBSS, spun down as before and resuspended in MEM 10% FBS. Viability and percentage of macrophages was measured as described above. Macrophages were then placed in 96 or 12-well cluster culture plates at a concentration of 3 X 10^ cells/cm^ and incubated at 37°C in a 5% CO2 atmosphere. Absence of mycoplasma contamination was ascertained by culturing the BAM without antibiotics, followed by microscopic examination after staining with the fluorescent dye 4',6'-diamidlno-2-phenylindole dihydrochloride (DAPI,. Calbiochem, San Diego,Calif.). Microorganisms. The SD-0 strain of M. dispar was isolated from a pneumonic calf (29), was cloned twice, and was used at passage level 7. Mycoplasma dispar was grown in Friis modified broth medium as previously described (19). Logarithmic phase cultures were centrifuged for 10 min at 500 X flf to remove debris and the resulting supernatant centrifuged at 34,000 X g for 30 min. The pellet was washed three times with 0.01 M phosphate buffered saline solution (PBS), pH 7.2. Protein concentrations were determined by the method of Lowry et al. (20 ) with alkali treatment prior to the assay to solubilize membranes (1 ). Mycoplasma suspensions were stored after quick freezing L 84b at -70°C in 1 ml aliquots (30 mg protein/ml). For induction of M. cf/spar capsule, we followed the methodology described by Almeida and Rosenbusch (3). Briefly, M. disparwere cocultured with bovine lung fibroblast monolayers for 23 h. Capsulated mycoplasmas were then harvested from the cell culture medium by differential centrifugation at 200 X g for 10 min at 4°C and washed 3 times by high speed centrifugation at 23,000 X g with cold PBS supplemented with 1 mM Tris and 1 mM EDTA (E-PBS). Aliquots of unencapsulated and encapsulated mycoplasmas were heat -killed by placing them in boiling water for 3 min. For use as a marker Serratia marcescens strain NIMA used in this experiment was grown on tryptose agar plates (Difco, Detroit, Mich.) and incubated at room temperature for 36 hours while Staphylococcus aureus (ATCC 10390) was grown in 7% bovine blood agar plates at 37°C overnight. The bacterial lawns were harvested and resuspended in lactose glutamate diluent (22) at a concentration of 6.0 X 10^ and 6.5 X 10^ colony forming units (CPU) per ml respectively, fractionated in 1 ml aliquots and kept at -70 °C until use. Capsule Purification. Capsule was extracted from the mycoplasmas by placing them in E-PBS (pH 8.0) at 37°C for 1 h. After centrifugation at 34,000 X g for 30 min, the supernatant was dialyzed for 72 h against the same buffer. Capsule material was then captured on immobilized Ricinus communis II lectin (Vector, Burlingame, Calif.), washed in decreasing salt concentrations (2 M, 1.1 M, and 0.2 M NaCI in 1 mM Tris ph 8.0 and 1 mM EDTA), eluted with 0.2 M lactose, and purified by size exclusion chromatography (Biogel P4, Bio-Pad, Richmond, Calif.) using E-PBS (pH 8.) as running buffer. Collected fractions were analyzed by thin layer chromatography (TLC) using TLC silica gel plates (E. Merck, Darmstadt, Germany) and a mixture of pyridine, ethanol, water, and n-butanol at a ratio of 4:1:1:1 as solvent system. Presence of capsule was detected by staining the plates with a saturated solution of silver nitrate diluted 1:10 in a mixture of H2O and acetone (1:1 ratio), followed by washing steps with 0.5 M NaOH in ethanol and then 6 M ammonium hydroxide solution. Fractions showing presence of capsule material were then analyzed following a radioimmunoprecipitation protocol previously described (3). Positive fractions were then pooled, lyophilized, resuspended at 15 ^ig/ml in 10 mM HEPES, 0.85 M NaCI and 0.1 mM CaClg buffer (pH 8.0), and kept in 500 p.1 aliquots at -70°C. Phagocytosis assay. The phagocytosis protocol described by Davis et al. (9) was followed. Briefly, 12-well cluster plates containing 3X10® /ml BAM were treated with 3X10® ccu/ml of encapsulated or unencapsulated M. dispaireaclBd previously for 10 min at 37°C with or without rabbit anti-capsule or anti-M. dispar sera at a 1:100 final dilution. Plates were then centrifuged at 800 X gr at 4°Cfor 20 min, and then incubated for 15 min at 37°C to promote mycoplasma attachment. Cultures were washed 5 times with warm MEM 5 % FBS to remove non-adherent mycoplasmas and incubated at 37°C in MEM with 10% FBS. At 4 h intervals after the addition of mycoplasmas macrophages of 3 wells per treatment were scraped from the plastic surface and centrifuged at 14,000 X g and 4°C for 10 min. The pellets were resuspended in MEM 10% FBS, sonicated on ice for 60 s (8 mm probe, 120 Watt; Braunsonic 1510, South San Francisco, Calif.) and the lysate was serially diluted 10-fold and plated in duplicate modified Friis plates (19) or in modified Friis broth tubes; broth tubes 86 and agar plates were incubated at 37°C in a 5% CO2 atmosphere for 5-7 days before titer determination or colony counting. Concomitantly with the last sampling, macrophage viability was checked in non-treated control wells by Trypan-Blue exclusion. Bactericidal Assay. Bovine alveolar macrophages in 12-well plates were exposed for 12 h to unencapsulated or encapsulated M. dispar, or to 7.5 |il/ml (final concentration) of purified capsule, or left untreated. Macrophages were then incubated for 4 h in MEM 10% FBS with 60 p.g/ml of tylosin (final concentration), washed 5 times with MEM 5% FBS and incubated with preopsonized Serratia marcescens or Staphylococcus aureus at 10:1 bacteria/BAM ratio. Plates were centrifuged at 800 X p for 5 min to assure contact between bacteria and BAM, incubated at 37°C for 2 h (S. marcescens) or 15 min (S. aureus) in a 5% CO2 atmosphere, and washed 5 times with warm MEM 5% PCS. The BAM cultures were then treated with warm MEM containing lysostaphin (1U/ ml) for 15 min (S. aureus), or 200 |ig/m of kanamycin and 100 |ig/ml of gentamycin for 2 h at 37°C (S. marcescens), followed by 5 washes with warm MEM 5%PBS to remove extracellular bacteria. Macrophages were then lysed with H2O and the number of viable intracellular bacteria were calculated by triplicate using end dilution titration in tryptose agar plates (S. marcescens) or in brain heart infusion agar plates (Difco, Detroit, Mi.) (S. aureus). Viability of BAM was checked in non-treated control wells by Trypan-Blue exclusion. TNF Assay. Bovine alveolar macrophages were treated with live or heat inactivated encapsulated or unencapsulated M. d/spar or treated with 7.5 ^g/ml 87a of purified capsule, 2 mg/ml Escherichia coli K235 endotoxin (Sigma, St. Louis, Mo), or culture medium, and incubated at 37°C in a 5% COg atmosphere. Each treatment was conducted in triplicate wells and ^0 p.1 samples of the supernatants were taken at 4, 8, 12, and 24 h after treatment. Samples were then centrifuged at 14,000 Xg for 10 min at 4°C, and kept at -70°C until use. Concomitantly with the last sampling, BAM viability was checked in non-treated control wells by Trypan-Blue exclusion. For the TNF assay, supernatants from the macrophages were assayed using the TNF sensitive L929 cell line. Briefly, L929 cells were grown in MEM with 10 % horse serum (HyClone, Logan Ut.), resuspended in the same media and placed in 96-well culture plates at a concentration of 2.4 XI0^ cells/well, and incubated at 37°C in a 5% CO2 atmosphere. The medium was then changed to MEM with 10% horse serum and 3 ng/ml of actinomycin D (Sigma, St. Louis, Mo.), and after this, wells were inoculated with 100 p,l of 1:10, 1:50 and 1:250 dilutions of macrophage supernatants, and incubated overnight at 37°C in a 5% CO2 atmosphere. After fixation with 10% formaldehyde, cells were washed 3 times with saline and stained with a 0.2% solution of crystal violet for 20 min. Plates were then washed to remove crystal violet, and dried for 24 h at 37°C. One hundred microliters of a 1:1 mixture of PBS (pH 7.2) and 100% ethanol was added to each well and then the plate was read at 595 nm in an automated ELISA reader (Bio-Tek Instruments Inc., Winooski, Vt). IL-1 Assay. Bovine alveolar macrophages were treated with live or heat killed encapsulated or unencapsulated M. d/spar or with 7.5 ng/ml of purified capsule, Escherichia coli K235 endotoxin (2 |ig/ml), or culture medium. Each 87b capsule, Escherichia coli K235 endotoxin (2 |xg/ml), or culture medium. Each treatment was conducted in triplicate wells and 300 |il samples of the supernatants were taken at 4, 8,12, and 24 h after treatment. With the last sampling, macrophage viability was checked in non-treated control wells by Trypan-Blue exclusion. Samples were then centrifuged at 14,000 X g for 10 min at 4°C, and kept at -70°C until use. For IL-1 activity, supernatants were assayed using the IL-1 dependent D10G4.1 cell line. Briefly, triplicate wells were inoculated with 1:5, 1:25 and 1:125 dilutions of macrophage supernatants in RPIy/ll 1640 suplemented with 5% PCS and incubated at 37°C in a 5% CO2 atmosphere for 48 h and then pulsed for 24 h with 50 |iCi/ml of - thymidine. Cells were then harvested on glass microfibre filters (Whatman, Maidstone, England) which were allowed to dry. Scintillation vials containing individual glass microfibre filters were filled with 2,5 ml of scintillation cocktail (Ready-Solv Hp/b, Beckman Instruments, Fullerton, Calif.) and counts per minute were determined in a scintillation counter (Packard Instruments, Downers Grove, II ). Glucose consumption. Ten microliter aliquots of supernatant from BAM treated with live or heat killed encapsulated or unencapsulated M. dispar, 7.5 |ig/ml purified capsule, or incubated with Escherichia coli K235 endotoxin (2^g/ml), or culture medium, were assayed for glucose consumption as indication of BAM activation, following the protocol described by Ryan et al. (24). Briefly, 10 ^il of each supernatant were mixed with 100 |il of a 5 mM NADP solution, 100 p.1 of a 20 mM ATP solution, and 5 ml of a hexokinase (250 units of hexokinase/ml) and G6PD mixture (Sigma, St. Louis, Mo), in 0.1 M Tris buffer ph 7.5. This mixture was added to a cuvette containing 700 p.1 of 0.1 M Tris-HCL [• 88 (pH 7.5), 64 mM NaCI, 3.5 mM MgCIa, and 0.15 mM CaClg. Two min after the solutions were mixed, the OD340 was read to determine NADPH production. Statistical Analysis. The data obtained from each experiment were analyzed by analysis of variance (ANOVA). When significant differences were found among groups, data were analyzed by paired f tests. Significance was accepted for comparison when the p value was less than 0.05. 89 RESULTS Phagocytosis. The results from the phagocytosis assay are shown In Fig 1A and B. When unencapsulated or encapsulated M.disparwere cultured with BAM in absence of antibodies specific for M. d/spar surface antigens, no reduction of the titer of the microorganisms was observed. The need for specific antibodies to promote phagocytosis was evidenced by a decrease of 2-3 logio in the titer of the mycoplasma. Bactericidal Activity. Bactericidal activity against opsonized S. aureus and S. marcescens was seen with BAM, as evidenced by reductions of 5 logs in the titer of these bacteria (Table 1). No effect on this bactericidal activity was observed when BAM were pre-treated with unencapsulated M. dispar. In contrast, inhibitory effect was detected when BAM were pre-treated with encapsulated mycoplasma since the titer of S. aureus or S. marcescens was over 2 logio higher in BAM treated with encapsulated M. disparthan that observed with the untreated macrophages. Similar results were observed when purified M. d/spar capsule was used to treat BAM prior to the assay. TNF Production. Live and heat killed unencapsulated M. dispar Induced marked TNF production that presents similar patterns of induction over time as that observed in endotoxin stimulated BAM (Fig 2A and 2B). Slight differences between live and heat-killed unencapsulated M. dispar were noticeable since levels of TNF production continued to increase up to 80 % of cytotoxicity at 20 h following the addition of heat-killed preparation while they reached 65 % of cytotoxicity with the live preparation in the same period of time. I: 90 When BAM were treated with live or heat killed encapsulated M. dispar, TNF production was significantly less (p< 0.05) than that of BAM stimulated with endotoxin or unencapsulated M. dispar. Likewise, purified capsule also had an inhibitory effect on TNF production by BAM. Fig. 1. Phagocytosis assay conducted with bovine alveolar macrophages: (A) unencapsulated M. dispar or (B) encapsulated M. dispar 'm presence and absence of specific antisemm against M.dispar or purified capsule. Symbols: unencapsulated M. d/spar without antibodies (•); unencapsulated M. dispar with anti-M d/sparantibodies (A); unencapsulated M. d/sparwith anti-capsule antibodies(#); encapsulated M. d/spar without antibodies (-•-); encapsulated M. d/sparwith anti-M d/spar antibodies {-A-y, encapsulated M. dispar with anti-capsule antibodies (-•-); Colony forming units (cfu). Mean of three samples are recorded with standard deviation of the mean. 8- Log cfu/ml O O ' '19' ' o- s- 93 Table 1. Killing of Serratia marcescens or Staphylococcus aureus by bovine alveolar macrophages pre-treated with unencapsulated M. dispar, encapsulated M. dispar or purified capsule. Pre-treatment None Microorganisms Unencapsulated Encapsulated Purified M. dispar M. dispar capsule S. marcescens 1.30®{1.02)b 1.48 (0.48) 3.70 (1.90) 3.02 (2.00) S. aureus 1.40(0.78) 1.54(0.90) 3.59(3.00) 3.30(2.95) ^Values are the mean of three counts expressed as log colony forming units/well. A total of 3 X 106 colony forming units of either S. marcescens or S. aureus was added to each well ^Values in parenthesis represent the standard deviation of the mean of three observations. IL-1 Production. Bovine alveolar macrophages produced lL-1 upon stimulation with unencapsulated M dispar, but the level reached was significantly lower than that observed with endotoxin stimulated BAM (Fig 3A). In contrast, the IL-1 production induced by encapsulated M. dispar or purified capsule was negligible and was no different from that detected with untreated macrophages. When heat killed encapsulated or unencapsulated M. dispar was used, it was observed that IL-1 was produced and at similar levels. Still, these were significantly lower than endotoxin stimulated macrophages but significant higher than untreated macrophages (Fig SB). r 94 Glucose Consumption. Glucose consumption was measured as an indicator of macrophage activation. In presence of unencapsulated M. disparthe rate of glucose consumption detected was similar to that observed in BAM stimulated with endotoxin (Fig. 4). In presence of encapsulated M. dispar, however, BAM showed only minimal consumption of glucose, significantly lower than that observed in endotoxin treated macrophages. Even more, when purified capsule was used, glucose consumption was less than the observed with encapsulated M. dispar and significantly different from that of BAM stimulated with endotoxin or treated with unencapsulated M dispar. 1 Fig 2. Production of tumor necrosis factor activity by bovine alveolar macrophages treated with live (A), or heat killed (B) encapsulated and unèncapsulated M. cf/spar Symbols: Media (A); untreated BAM (O); endotoxin (•); purified capsule (•); encapsulated M. dispar{xy, heat killed capsulated M. dispar, (-x-); live unencapsulated M.dispar {%)•, heat killed unencapsulated M. dispar 96 (A) (B) 100 100 80 > 60 u 60 M e >> u 40 20 "O 0 10 TIm# (h) 20 30 20 10 Tlm*(h) 30 Fig. 3. Production of interleukin-1 {IL-1) by bovine alveolar macrophages (BAM) treated with live (A), or heat killed (B) encapsulated and unencapsulated M. dispar. Symbols as in Fig.2 98 (B) 40000 1 40000 1 30000 - 30000 & & o S • Z 1 • 20000 - m" 20000 10000 10000 - —I T 10 20 TIm# In hour* 10 1— 20 TIm# In hours —I 30 Fig 4. Glucose consumption of bovine alveolar macrophages treated with unencapsulated M. dispar, encapsulated M. dispar or purified capsule. Symbols: endotoxin (•); unencapsulated M. dispar (9), encapsulated M. dispar {xy, purified capsule (•), untreated BAM 100 100 1 80 - at c E 2 I t» o 20 40 60 80 Time (h) 100 120 140 101 DISCUSSION Pathogenic microorganisms have evolved several strategies to avoid phagocytosis by interfering at distinct steps of the attachment, ingestion, and killing process. Among these, microbial capsules were considered the most important and ubiquitous anti-phagocytic substance (15). Some microorganisms posses surface structures that enable them to attach to phagocytes without being ingested. Neisseria gonorrheae and several mycoplasmas have been reported among these microorganisms and in N. gonorrheae such property was linked to their capsule (10). While it is not known if the capsule of M. dispar p\ays a role in attachment, it has been shown that capsule production occurs in vivo or in coculture with bovine cells (3). The results presented in this report indicated that the capsule of M dispar exerts antiphagocytic activity. When encapsulated or unencapsulated M. dispar were exposed to alveolar macrophages in absence of specific antibodies, no loss of viability of the mycoplasma was detected. In contrast, in presence of antibodies specific to capsule or surface proteins, significant loss of viability to encapsulated or unencapsulated M. dispar, respectively, was detected. The protective role of the capsule can be deduced by the fact that complete loss of viability of the mycoplasma was detected only when encapsulated mycoplasmas were exposed to BAM in presence of specific anti-capsule antibodies. The failure to obtain the same reduction in mycoplasma viability observed in the system which included unencapsulated mycoplasmas BAM and M. d/spar antiserum, can be explained by the persistence of a low percentage of encapsulated M dispar \n unencapsulated mycoplasma grown in broth media (3). 102 This fraction of the mycoplasma population will resist phagocytosis in absence of specific anti-capsule antibodies. It was previously reported that mycoplasmas can inhibit the phagocytosis and killing of other microorganisms (17, 26, 28). It was postulated that this effect was independent of the attachment of the mycoplasmas to the phagocyte membrane and this was supported by the finding that Mycoplasma arthritidis did not attached to cells but still inhibited phagocytosis (17). Also, It was reported that the capsular polysaccharide of Pseudomona aeruginosa Inhibited phagocytosis by blocking mannose receptors on the surface of the macrophages (6). The results presented here are in agreement with these previous reports. Capsulated M. dispar or purified capsule inhibited the killing of S.maKescens or S.aureus by 2 logs compared to the killing activity exhibited by untreated BAM or BAM treated with unencapsulated M. dispar. From these results it can be concluded that the presence of capsule, as a purified substance or in association with M. dispar cell surfaces was linked with a diminished bactericidal activity of the BAM. A previous report showed that M. dispar exerts a immunosuppressive effect when injected in combination with other antigens (16). It also was reported that M. dispar exerts an inhibitory effect on the mixed lymphocyte response to mitogens. It also was suggested that this suppressive effect coukj be related to the avidity with which this mycoplasma species adheres to the host cell membrane, resulting in alteration in the membrane /receptor structures on the host cell and the production of a suppressive factor (11,16). In the present report we demonstrate that encapsulated M. dispar or purified capsule did not induce TNF or IL-1 production by BAM, in contrast to what was seen when unencapsulated M. dispar v/as used. In addition, encapsulated mycoplasma or 103 purified capsule did not Induce glucose consumption by BAM, indicating that these cells were not activated. In contrast, activation of macrophages was observed when macrophages were incubated with unencapsulated M. dispar or with endotoxin. It is possible that the lack of activation of BAM treated with purified capsule, only reflects the inability of this material to interact with macrophages, but the reduced bactericidal activity observed in BAM cultured with purified capsule, indicates that a mechanism of inhibition was operative. It was observed that encapsulated M. d/spar induced slightly higher levels of TNF production and glucose consumption compared with that of purified capsule. These differences can be explained by the small percentage of unencapsulated mycoplasmas that are detected when expression of mycoplasma capsule was induced by coculture (3). It also was shown that IIL-1 production by BAM stimulated with heat-killed encapsulated mycoplasmas was significantly higher than that observed with live encapsulated M. dispar. It is possible that during the process of heat-killing, capsule material was lost from the surface of the mycoplasma unmasking regions of the membrane that couki react with the IL-1 receptor in the macrophage cell surface. It also was possible that fractions of disrupted membrane, released during heat killing, could react with BAM causing IL-1 production. These differences were not detected with TNF production, which indicated that TNF and IL-1 induction in BAM could be dissociated. The inhibitory effect of encapsulated mycoplasmas or capsular material on the induction of TNF and IL-1 production or glucose consumption, indicated that this material was blocking the activation of the macrophages. It was previously shown with other mycoplasma species that induction of TNF production resulted r_. 104 from the interaction of a mycoplasmal membrane lipoprotein with the surface of the macrophage (25). It is possible therefore, that the capsule material of M. d/spar could mask a similar lipoprotein or its receptor in the surface of the macrophage, abrogating their activation. It was previously shown that Mycoplasma mycoides subsp. mycoides (LC) induced TNF production, and that injection of TNF in goats reproduced the disease caused by this mycoplasma species (23). It also was postulated that Mycoplasma bovis may induce production of cytokines and the effect of these could explain in part the necrotic lesions and lymphocyte accumulations detected in the lung of naturally infected calves (11). In the present report we show that encapsulated M. dispar diô not induce production of IL-1 and TNF or glucose consumption by BAM. The lack of macrophage activation coukJ explain the poor immunogenic reactivity induced by M. dispar and also the characteristic interstitial alveolitis without peribronchial lymphocyte accumulations caused by this mycoplasma species (13,16). The inhibitory effect exerted by the capsular material or encapsulated M. dispar on killing capacity of the BAM to secondary bacteria and the diminished activation and secretion of cytokines, defines the important influence that the infection by M. dispar may have on the development of severe pneumonic processes. We are aware of the fact that these results describe the interaction of M. d/spar with BAM under in vitro conditions, and these may not completely reflect the in vivo situation. However, the immunosuppressive effects previously reported (16) and the results presented here, can be taken together to help define the role that M. dispar has on the development of pneumonic conditions in cattle. 105 REFERENCES 1. Albro, P. W. 1975. Determination of protein in preparations of microsomes. Anal. Biochem. 64:485-493 2. Al-Kaissi, A., and M. R. Aley. 1983. Electron microscopic studies of the interaction between ovine alveolar macrophages and Mycoplasma ovipneumoniae in vitro. Vet. 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Assoc. 175:812-813. 110 GENERAL CONCLUSIONS The main objective of the present work was to elucidate the effect that the infection with Mycoplasma cf/spar infection exerts on the activity of two main components of the defense mechanism of the bovine respiratory system (e.g., tracheobronchial clearance and alveolar macrophage). In the first manuscript, the focus was to determine the effect that M. dispar has on the mucociliary activity of calves. The approach used was to compare the tracheobronchial elimination of a bacterial marker {Serratia marcescens) before and after the infection with M. dispar on the same calves. In order to assure that inoculation with the bacterial marker does not in itself influence their rate of elimination, the tracheobronchial elimination of the bacterial marker in calves non-infected with M. dispar was determined using the same sampling routine and route frequency of inoculation. It was found that before the infection with M. dispar \he elimination showed a peak around 2 hours after inoculation with the marker, and by 4 hours only a few colonies were detected. In contrast, after infection with M. dispar, the rate of elimination was steady and by 4 hours after inoculation with the marker, the titer of the bacterial marker was 4 logs higher than that detected in the preinfection status calves. In addition, the bacterial marker was isolated in 83% of the lungs from to the Md/spar infected group, compared with no isolation in the non-mycoplasma infected calves. This can be taken as indication that the clearance of the marker was incomplete 24 hours later in the M. dispar inoculated group of calves, which was in contrast with that observed in the nonmycoplasma inoculated group of calves. I' 111 The study was also extended to include histopathology and scanning electron microscopic examination of the lung tissues. The main lesion observed centred in the small and medium size airways . In these, the ciliated ephitelium presented sign of degenration with shinkage of the cytoplasm, pyknosis and sloughing of the cell. The lumen on'these airways contained PMN's and mononuclear cells, and a moderate infiltration of PMN's and macrophages were seen in the submucosa.. The SEM observation of these airways showed loss of ciliated epithelial cells with patching distribution and presence of inflammatory cells along the mucosa of small airways. No lesions of the respiratory epithelium was detected in the large airways and no lesions were detected in the lungs of the calves not exposed to M. dispar The results obtained in this part of the project indicated that the infection with M. dispar was associated with a reduction in the tracheal elimination of the bacterial marker. This reduction was also associated with damage to the ciliated epithelium of small airways, which was observed only in the M. dispar exposed calves. The second part of the project was to assess the effect that M. dispar infection exerts on bovine alveolar macrophages (BAM). The approach used was the evaluation of phagocytic functions, secretory functions, and activation capacity of BAM infected with encapsulated, unencapsulated M. dispar and purified capsule. It was found that phagocytosis did occur when unencapsulated or encapsulated M. dispar was exposed to BAM in absence of specific antibodies. The same results were observed when encapsulated M. dispar was exposed to BAM in presence of antiserum against unencapsulated 112 M.dispar, or when unencapsulated mycoplasmas were exposed to BAM In presence of antl-capsule antibodies. In contrast, reduction in the titer of the mycoplasma was detected when unencapsulated mycoplasmas were incubated with BAM in presence of specific antiserum, but complete reduction in the titer was detected when encapsulated M. dispar was incubated with macrophages in presence of specific anti-capsule antibodies. These results indicated that the capsule exerts an anti-phagocytic effect, and that this effect can be overcome by the addition of specific anti-capsule antibodies. The anti phagocytic effect of microbial capsules is not a novel discovery, since the same effect was described long ago in many pathogenic microorganisms, but what was interesting in the present work, was the finding that the encapsulated mycoplasma or the purified capsular material also inhibited the killing of other microorganisms added to the system. In effect, when macrophages were pretreated with encapsulated M. dispar or purified capsule and subsequently exposed to Staphyloccocus aureus or Serratia marcescens in presence of specific antibodies, a significant reduction of the killing activity of the macrophages was detected, compared with that observed in non-mycoplasma or capsule treated BAM. This effect was not detected when BAM were pretreated with unencapsulated M dispar. It was also shown that encapsulated M. dispar or purified capsular material did not emhance glucose consumption, production of TNF, or IL-1. In contrast, when BAM were treated with unencapsulated M. dispar, increase in glucose consumption, and production of TNF as well as IL-1 was observed. However, some differences were observed when these results were compared with that of heat-inactivated encapsulated or non-encapsulated M. dispar. For 113 instance, IL-1 production by BAM stimulated with heat-inactivated encapsulated mycoplasmas was significant higher than that observed with live encapsulated M. dispar. A possible explanation is that the heat-inactivation caused the release of the capsule material from the surface of the mycoplasma leaving exposed regions of the membrane that could react with the macrophage cell surface. It is also possible that fractions of disrupted membrane, released during this process, could react with BAM causing IL-1 production. These differences were not detected with TNF production or glucose consumption, which indicated that the inhibition of these functions operates by a different mechanism . It was previously shown with other mycoplasma species that induction of TNF resulted from the interaction of a mycoplasmal membrane lipoprotein with the surface of the macrophage (Sher et al, 1990). It is possible therefore, that the capsule material of M. cf/spar could mask a similar lipoprotein or its receptor in the surface of the macrophage, avoiding their activation. However, this can not explain the inhibitory effect exerted by the capsular material to the killing activity of BAM to a second microorganism such as S. marcescens or S. aureus or the inhibitory effect exerted by the capsule on the activation of the macrophages. This indicates that the action of the capsular material is not simply mechanical by masking the surface of the mycoplasmas, but also by inhibiting macrophage functions. 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Immunology of the lung and upper respiratory tract. New York, McGrawHill. r 140 ACKNOWLEDGEMENTS I wish to express my gratitude to my major professor Dr. Ricardo F.Rosenbusch and members of my PhD committee for their patience, guidance, support, and contribution during the execution of the present thesis project. I extend my thanks to the people in our laboratory for their support and technical assistance. A very special appreciation is directed to my wife Eugenia Graciela and our four kids, Camila Otilia, Antonio Mariano, Soledad, and Juan Manuel for their continuous sacrifices, trust, and support given through this thesis wori<.