* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Role and significance of the complement system in mucosal
Survey
Document related concepts
Monoclonal antibody wikipedia , lookup
Hygiene hypothesis wikipedia , lookup
Molecular mimicry wikipedia , lookup
Adaptive immune system wikipedia , lookup
Immune system wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Innate immune system wikipedia , lookup
Immunosuppressive drug wikipedia , lookup
Transcript
Immunology and Cell Biology (2001) 79, 1–10 Review Article Role and significance of the complement system in mucosal immunity: Particular reference to the human breast milk complement MICHAEL O OGUNDELE Department of Medical Informatics, University of Applied Sciences, Berlin, Germany Summary The complement system plays an important role in a host’s defence mechanisms, such as in immune bacteriolysis, neutralization of viruses, immune adherence, immunoconglutination and in enhancement of phagocytosis. The possible role of this important biological system in biological fluids on the mucosal surfaces, including breast milk, has however been largely neglected. Its contribution to the ‘common’ mucosal immunity is still enigmatic and largely speculative. Assessment of the complement system in human breast milk, which has so far largely been limited to different assays of the individual component proteins, is reviewed. A brief review of the classical and the alternative pathways of complement activation is presented. The potential physiological roles of various complement components and their activation fragments in human milk in particular, and other mucosal surfaces in general, are also presented. It was concluded that the complement system might play a complementary role to other immunological and non-immunological protective mechanisms on the mucosal surfaces. Key words: allergy, bacteriolysis, complement system, human breast milk, infection, mucosal immunity. Introduction The mucosal surfaces of the body play a vital role in the interaction between the external and internal environments. They must be able to simultaneously protect the underlying tissues against the onslaught of infectious agents and regulate the uptake of useful environmental agents, such as food antigens. They have therefore evolved peculiar immunological mechanisms for performing their multifaceted functions. Mucosal immunity as a common functional entity has been fairly well defined, at least for B lymphocytes and their precursors, and recently for T cells, which are located in all mucosal tissues.1,2 These concepts have led to advanced knowledge in the study of cellular mechanisms of immunity on the mucosal surfaces and applying such a hypothesis for the humoral defence mechanisms may lead to much more desirable progress in this field of study also. There is a conclusive body of evidence that breast-feeding protects the infant against a wide range of infectious and other diseases.3,4 Efforts have been directed in the past few years to identify various immune-active substances in human breast milk (HBM), which account for the observed protective effects. These include specific antiviral antibodies,5 specific antibacterial antibodies,6 IgG, IgA, IgM,7–9 lactoferrin,9 transferrin, lactoperoxidases, and lactose, which increases calcium absorption and promotes growth of Lactobacillus bifidus and prevents gut colonization by pathogenic organisms, lysozyme10 different cytokines,11 lymphocytes, Correspondence: Dr MO Ogundele, Department of Medical Informatics, University of Applied Sciences, Foehrer Str. 6, D-13353 Berlin, Germany. Email: [email protected] Received 2 March 2000; accepted 7 August 2000. polymorphonuclear leucocytes and macrophages.12 Human milk contains antiproteases, which protect biologically active proteins from enzymatic destruction in the mammary gland and in the infantile gut. Human milk also contains digestive enzymes, lipases, which assist in ensuring optimal nutrient assimilation, and various hormones, including thyroidstimulating hormone (TSH), thyroid release-stimulating hormone (TRH), cAMP, triiodothyronine (T3) and thyroxine (T4), erythropoietin, growth release-stimulating hormone (GRH), prolactin, corticosteroid-binding protein and growth promoting agents, epidermal growth factor (EGF) and neuroepithelial growth factor (NGF).13 The complement system was first discovered in the serum by Jules Bordet in 1894 as a heat-labile factor that facilitated the killing of bacteria by specific antibodies. The complement system plays an important role in the host defence mechanisms, such as in immune bacteriolysis, immune adherence, immunoconglutination and in enhancement of phagocytosis.14 The complement system is one of the earliest systems to be fully established in mucosal tissues during the neonatal period.15 The serum complement system consists of at least 19 proteins, mostly in pre-activated enzymatic forms, activated in a multistep cascade reaction via the classical or alternative pathways. Other less prominent activation pathways also exist, including the mannan-binding protein (lectin) and C-reactive protein pathways. The classical pathway is activated mainly by antigen–antibody complexes (IgG or IgM mostly) starting with C1q.16 The alternative pathway (APC) utilizes active sites (that are present on zymosan, yeast, cobra venom, gram-negative bacteria, sheep erythrocytes and human cells deficient in the expression of regulatory molecules) in the presence of properdin, serum factors B and D, 2 MO Ogundele to activate C3. The two pathways proceed uniformly after C3 activation to the formation of (C5b-9) membrane attack complexes (MAC), capable of inserting into biological membranes and producing cell lysis and death.17 The possible role of this important biological system in breast milk and in other mucosal secretions has, however, been largely neglected. Assessment of the complement system in HBM has been largely limited so far to assays of the individual component proteins (Tables 1 and 2). The relatively low levels of most of the complement components in mature milk (as opposed to the levels in colostrum and transitional milk), have been cited as an argument against any significant physiological activity of the complement system in breast milk.18 Human milk also contains a wide range of anti-inflammatory factors, which are capable of inhibiting complement activation.19 Moreover, the application of standard methods for assessing the serum complement are Table 1 unreliable in studies of breast milk complement.20 These and many other problems associated with handling human milk samples have hampered further research into the possible contribution of the milk complement to the observed protection of the nursing infant against many infectious diseases. These problems are part of the reason why emphasis has been placed on the pro-inflammatory effects of the complement, which have been observed to be minimal in the breast milk, to the neglect of its other potential immunomodulating and protective activities. Immunochemical levels of breast milk complement components Using radial immunodiffusion assays, levels of C3 and C4 comparable to those in normal serum have been found in early colostrum. While the level of C4 fell precipitously from Immunochemical levels of major complement components and related proteins in human breast milk and serum Component Serum level (µg/mL) Colostrum level (µg/mL) C1q C3 126 ± 1.0* 1048 ± 209 † 1830 1640 ± 110* 19*§ 15 110 ‡ 6 1081 ± 17.0* 450 ± 50.0* 213 ± 64† 530 27.1* 442.0 ± 35* 140 ± 35* 102.66 † 319 ± 74 810 1050 ± 270 ng/mL† 702 ± 292 † C4 C7 B D H Serum level (%) Mature milk level (µg/mL) 115 ± 9.5* 12.5–19.3* 160–170 250–330 173.6 ± 16.9* 674.0 ± 363* 16 150 50 –* Serum level (%) 7.0 Nil 9.4 289 ± 242* 206 20 ‡ 16.03 ng/mL† 3.51 † 2.5 1.8 0.5 11.3 ± 33* 52.0 ± 7.6* 20* 570 ng/mL† 11.8 14.0 0.07 4.20 ng/mL† 0.72 † 0.4 0.1 Reference 27 29,32 22 25 8 23 27 21 29 22 8 25 21 29 29 22 Unpubl. Unpubl. *Radial immunodiffusion (RID); †enzyme-linked immunosorbent assay; ‡10 000 g centrifugation rate; §RID after protein precipitation to achieve a concentration 63-fold higher than normal colostrum samples; Unpubl., MO Ogundele, unpubl. obs., 1998. Table 2 Haemolytic titration levels (haemolytic units) of major complement components in human breast milk and serum Component C1 C2 C3 C4 C5 C6 C7 C8 C9 Factor B Serum level Colostrum level Serum level (%) Centrifugation (g) Reference 24–32 000 76.9 2400–4800 55.9 molecules/mL 3200–9600 14.1 molecules/mL 128–512 000 526 molecules/mL 32–48 000 103 molecules/mL 96–128 000 240–320 000 60–80 000 60–120 000 63 molecules/mL 9–21 0.03 22 000 2 0.2 0.8 2 5 2.5 0.3 0.007 0.3 7 0.5 6 1.2 22 000 10 000 22 000 10 000 22 000 10 000 22 000 10 000 22 000 22 000 22 000 22 000 28 22 28 22 28 22 28 22 28 22 28 28 28 28 22 3–160 0.094 molecules/mL 20–67 0.25 molecules/mL 4700–12 000 13.0 molecules/mL 100–150 0.007 molecules/mL 260–380 20–27 000 240–320 4400–7600 0.77 molecules/mL Roles of the mucosal complement system approximately 0.18 mg/mL to 0.01 mg/mL during the first month of lactation, no C3 was detectable after 6 days of lactation.21 Other studies have, however, shown relatively stable and low levels of C3 and C4 in mature milk for up to 18 months of lactation.22–25 C1 and C5 could not be detected immunochemically in the colostrum by Ballow et al., who conducted one of the earliest studies on breast milk complement.26 By using a high concentration technique, C1q was later detected in human colostrum.27 Table 1 shows up to a 5-fold difference between the immunochemical levels of C3 and C4 as reported by various authors, probably reflecting inherent variability of these proteins in breast milk among different populations, but this variation may also reflect differences arising from different assay methods. These results emphasize the need for a standard assay of these proteins and the need to obtain a simultaneous serum level to enable comparisons between different studies. The level of factor B that has been measured immunochemically averages 2.5% of normal human serum.22 An approximate level of 15–20% C3 proactivator (factor B) functional activity has been detected in the colostrum when compared with normal human serum.26 Evidence for an intact alternative pathway of complement activity in breast milk, however, suggests that factor D is also present in physiologically significant levels.28 Haemolytic titration of colostral complement components Initial studies of the haemolytic activity of breast milk complement were carried out on colostrum that was stored for 24 h at 0°C, and no haemolytic activity of C3 or C4 was detectable. Evidence for the presence of activated fragments of C3 and C4 has, however, been obtained through gel electrophoresis and immune adherence assays.26 Subsequent studies by Nakajima et al. have shown the presence of haemolytic activity of all nine components of the classical complement in colostrum, ranging from 0.03 to 7% that of normal human serum.28 Evidence for the presence of factors of the alternative pathway has also been obtained. C4, C7 and C9 demonstrate relatively high haemolytic activities, while C1 shows the lowest activity.28 Haemolytic assay of breast milk complement activity A haemolytic assay based on a micromodification of the standard 50% complement haemolysis (CH50) test for the classical and alternative pathways of human breast milk complement has recently been developed. Using this assay, the haemolytic activity of human milk and colostrum was found to be between 0 and 18.5 CH50/mL, compared with values of 25–45 CH50/mL for normal human serum (pers. obs.). This is comparable with levels of 6.25–12.5% serum complement haemolytic activity reported in normal human tears, which is another site of mucosal immunity.34 An assessment of the overall haemolytic activity of the HBM complement has been hampered, in part, by the relatively small amount of the component present in mature HBM compared with the serum and therefore the need to develop a more sensitive assay technique. Further hindrances 3 to the development of these assays are the recognizable inhibitory or ‘anti-complement’ factors present in breast milk.19 This inhibitory effect in bovine milk has been, in part, ascribed to the prozone phenomenon of excessive antibodies in undiluted milk, and other unexplained factors in heated milk, particularly in the casein micelles.35 Cole et al. have also discovered that some complement components in human milk may be haemolytically inactive, although physiochemically innate and immunochemically detectable.22 The high lipid content in colostrum also physically interfers with the optical density measurements in the complement assays.36 Furthermore, there is apparently a relative deficiency in some of the essential components of the complement cascade system. For example, properdin, a stabilizer of fluid-phase alternative pathway convertases, has been reported to be either absent in human breast milk, or only present in minute quantities < 1 µg/mL.37 Local synthesis versus systemic source of mucosal complement More than 90% of the detectable serum C3 and C4 have been shown, through allotype conversion following hepatic transplantation, to be produced by the liver.38 Most of the other body tissues are also able to synthesize all the components of the complement system, including milk macrophages. Evidence for the local synthesis of complement components has been obtained in virtually all organs involved in mucosal immunity, both by normal tissue and in various pathological conditions: kidney, intestinal and conjuctival mucosa. This local complement synthesis may even predominate in some tissues.22,39–43 Local complement synthesis is regulated by locally generated cytokines, suggesting a real significance in the physiological and pathological conditions of the tissues.44 The presence of cytokines in human milk would tend to favour local synthesis of the complement by breast milk macrophages.11 It is also possible that the mammary gland epithelial cells are at least partly involved in local complement synthesis, as is observed in intestinal mucosal cells. It is yet to be clarified in HBM what relative contribution of the complement is transported from the serum, as well as the role and the control of the local synthesis. A major source of locally synthesized complement, in addition to the tissue cells, are tissue macrophages and blood-derived monocytes. The rate of secretion of C2 and factor B by milk macrophages has been found to be eightfold and 2.5-fold, respectively that of blood monocytes. C2 and factor B produced by milk macrophages also constituted a five- to 16-fold greater proportion of total protein synthesis compared with blood monocytes.22 Pathways of mucosal complement activation Classical versus alternative pathway of human breast milk activation in vivo The serum complement system cascade reaction occurs via either the classical or alternative pathway. The levels of C1q in breast milk are extremely low,27 while levels of IgG and IgM are moderately low, in comparison to their respective serum levels7–9 (Table 3). Secretory IgA (sIgA) is the most 4 MO Ogundele Table 3 Levels of major proteins and cations related to complement activation in human breast milk and serum Component Serum level (mg/dL) Magnesium 1.87–2.51 Calcium 3.16 ± 0.41‡ 8.8–10.6‡§ IgA IgG IgM All proteins Albumin †† Colostrum level (mg/dL) Serum level (%) 9.84 ± 0.17‡ 1.20–4.50 g/L¶ 1.69 ± 0.25 g/L†† 2.02 ± 56 g/L†† 2767 ± 194 †† 1188 ± 240†† 100–240 258 ± 27†† 147 ± 50†† 7.76 g/L 3.36 ± 0.37 g/L 2.1 ± 2.3 g/L 118 74 87.93 ± 47 g/L†† 4300 5.9 ± 1.58 0.2 48.9 ± 13.8†† 17.1 ± 4.29†† 4.1 10 177.4 ± 113†† 120 100–150 mg/mL* 82 mg/mL†† 1600 1000 6.4 ± 0.10 mg/mL* 4 g/dL 4.5 g/dL†† ~0.18 g/dL†† 4 Mature milk level (mg/dL) ~4‡ 4.9 ± 0.1 4.67 ± 0.15‡ 11.9–46.9‡§ 26.2 ± 0.5 23.6 ± 0.35‡ 32.3–45.6 ± 2.7** 1.2 ± 0.08 g/L 1.0 ± 0.5 g/L 0.29 ± 0.014 g/L†† 1.50–4.9 g/L†† 55 ± 7†† 2.9 ± 0.92 5.37–6.49 ~5.0†† 2.9 ± 0.92 2.81–4.59 24.8 ± 6†† ~10†† 30–40 mg/mL* 20 mg/mL†† 7.09–8.39 mg/mL ~35†† Serum level (%) 183 224 148 303 270 240 42 35 17 74–243 2 0.1 0.2 0.4 1.7 2.2 10 6.8 450 250 120 0.8 Reference 30 33 31 8,30 33 31 8 24 20 25 21 25 24 8 21 24 8 25 21 30 30 21 31 30 21 *Biuret protein analysis method; †female population; ‡atom-absorptions-spectroscopy; §flame photometry; ¶nephelometry; **arbitrary unit; radial immunodiffusion; ~, approximated value. abundant immunoglobulin in colostrum and breast milk, as well as in most other secretory body fluids. Immunoglobulin A is known to be a poor activator of the alternative pathway and an inhibitor of the IgG-mediated classical pathway of complement activation.45,46 The other components of the classical pathway of complement activity (C2 and C4) are immunochemically present at a relatively higher level than the components of the alternative pathway (factor B and factor D). C1q, however, seems to be the limiting factor for the classical pathway in breast milk, based on observations of bovine milk.47 This would suggest that the alternative pathway may predominate in human breast milk, compared to the classical pathway. The observation that C3 binds to renal proximal tubules in the absence of C1q or C4 also supports the hypothesis that the complement system may be activated by the alternative pathway using the brush border of the renal epithelial cells.48 Non-immune complement activation Increased levels of C3a anaphylatoxin (AT) peptide in tears of patients with conjunctivitis in the absence of immune complexes has been taken to suggest non-immune generation.49,50 The presence of a number of proteases in HBM may contribute to the non-immune generation of AT peptides.51 Breast milk proteinases are capable of cleaving casein and other biologically active proteins, probably also including native complement precursors of AT peptides. These proteinases have been shown to be heat resistant and to occur in higher levels in mastitis, being active in both whole and skim milk.52 During the passage of breast milk complement through the intestinal tract, partial digestion of native proteins may lead to the non-immune production of AT peptides, which may have significant physiological roles in the intestinal tract. Anaphylatoxin peptides are particularly resistant to chemical denaturation and acidification and may be able to retain their physiological functions throughout the length of the intestine. These functions may include regulation of immunological reactions and bowel motion. Proposed physiological mechanisms of mucosal complement activation There is a wide variety of humoral factors in HBM and other mucosal secretions, which inhibit the optimal activity of the complement system.19,53,54 Activation of the complement may, therefore, be expected to preferentially take place on any available activating surface rather than in the fluid phase, thereby favouring the alternative pathway of activation.17 In vivo, milk fat globule membrane (MFGM) in human milk and luminal surfaces of epithelial cells in other mucosal surfaces may serve as an abundant supply of a suitable template for complement activation, where all the products of activation are released in situ to obtain a relatively high local concentration and maximal effect on the membrane-bound antigens. Roles of the mucosal complement system In human milk, MFGM has been shown to adhere closely to certain pathogenic bacteria through their surface glycoproteins, thereby preventing the colonization and infection of the buccal epithelial cells in the oral cavity of a suckling neonate.55 The MFGM and other activating surfaces provide a suitable environment for the attached antigens to come into close contact with native and activated complement components. These activating surfaces may assume particular significance when the level of potential pathogens overwhelms the inhibitory ability of the sIgA and the other secretory bacteriostatic components of the different body fluids. The pathogens may then be bound to the activating surfaces where they could activate the complement system and subsequently be killed by lysis, without significant interference from the diverse complement inhibitors present in the fluid phase. Homologous cells are protected from the lytic effect of complement through the expression of surface membrane regulatory molecules, such as decay accelerating factors (DAF, CD55), membrane cofactor protein (MCP), homologous inhibitor of reactive lysis (protectin, CD59), C3 receptors 1, 3 and 4 (CD35, CD11b, c/18). Active forms of protectin have been found on MFGM as well as soluble forms in breast milk.56 Decay accelerating factor has been detected on most epithelial cells at sites of mucosal immunity as well as in soluble form in various body fluids.57,58 Vitronectin is another complement regulatory molecule that has been detected in human tears.59 Because these regulatory molecules are physiologically essential in protecting body cells from autologous complement attack, their identification on several mucosal sites, the ocular surface, intraocularly, the lacrimal gland, in tears, mammary glands and in human milk suggests that physiological control of complement activation is also required in these locations. The mucosal surfaces constitute strategic host barriers against a continual invasion by organisms both of the normal body flora and environmental pathogens, where the complement system and other defence factors are constantly being activated. The epithelial cells therefore need to express these regulatory molecules to protect them against the lytic effects of the ongoing complement activation. Physiological significance of mucosal complement Mucosal complement secretion and activities are known to increase in states of infection or other inflammatory disorders of the mucosal membranes, including mastitis in the mammary gland, microbial corneal ulcers and conjunctivitis in the eye, chronic inflammatory bowel diseases of the intestine and inflammatory nephritides.34,42,43,49,50 A deficient production of local complement components has been associated with an increased risk of developing mastitis in lactating mothers.60 Skin sepsis in the suckling infant is also associated with an increased level of complement secretion in breast milk.7 Although these conditions do not necessarily prove any cause–effect relationship, they do point to the possible physiological role of the complement system on mucosal surfaces. They also suggest that the mucosal complement system is physiologically induced by disease states and is therefore closely regulated in health by 5 some natural mechanisms and body components, and may therefore contribute to the protection of the host at these mucosal sites. Possible roles and functions of mucosal complement Bacterial opsonization In vitro activation of the complement system and subsequent deposition of C3 opsonin fragments on solid-phase killed bacteria have been documented in both bovine and human milk.47,61 Studies have previously shown that bacteria coated with C3 fragments can be ingested by phagocytic cells in the absence of antibodies, even when the cells are in the resting state.62 The complement system therefore probably constitutes a vital component of mucosal immunity with respect to opsonization and phagocytosis of pathogens. C3 fragments are also known to greatly enhance the opsonizing effects of IgG antibodies more than 100-fold. Optimal phagocytosis and killing of bacteria depend on the synergistic actions of both the complement and the antibodies acting through their respective receptors on the membrane of the phagocytic cells.62 The complement receptors, which participate in the phagocytosis of foreign antigens, are known to be upregulated in conditions of inflammation or complement activation, in the presence of such mediators as C5a.63 Bactericidal activities The bactericidal activities of serum complement have been well established.64 Different strains of bacteria are known to be differently sensitive to the lytic effects of complement. Bacteria can be killed by any of the four mechanisms of complement activation, namely, direct and antibody-dependent classical pathway, and direct and antibody-dependent alternative pathway activation.65 Direct complement-induced bactericidal activities have also been detected in human milk and colostrum,61 as well as in bovine colostrum and mastitic milk.66 These findings have suggested that the mucosal complement system constitutes an important defence factor against local infection of the mammary gland by foreign pathogens. The observation that the bacterial count in breast milk decreases during in vitro storage at 4°C suggested that an antimicrobial process is activated during this period.67 In addition to the lipolysis-induced cytolytic effects, other complement-dependent mechanisms could also be responsible. The presence of contaminating microorganisms and abundant IgA could activate the alternative pathway of the complement system at this temperature.17 Certain strains of Streptococcus have been shown to be capable of directly activating the serum and bovine milk complement.47 Furthermore, specific IgG directed against virulence factors of bacteria are present in human milk and other mucosal secretions.6 It is conceivable that exposure to these bacteria in the gastrointestinal tract of the infant could lead to the activation of the classical pathway of complement and could assist in the elimination of these organisms, thus preventing colonization or infection. The bactericidal effect of bovine milk colostrum and specific antibodies have been suggested to account for the favourable influence of breastfeeding on gut colonization.68 6 MO Ogundele Haemolytic activity of complement has been shown to be higher in bovine mastitic milk and this increased haemolytic activity is correlated with an increased level of bacteriolysis.68 Increased levels of C3 and C4 have also been demonstrated in human mastitic milk, although the functional activity of this elevated level has not been determined.60 Cytotoxicity against virus-infected cells Protection of the breast-fed infant against maternal–infant transmission of viral diseases including HIV probably involves the activity of the breast milk complement. The lack of persistence of IgA and IgM, both of which can activate complement-mediated cell lysis in the presence of viralinfected cells, has been found to be strongly associated with increased risks of transmitting HIV-1 infection to the infant.69 Viral infections at mucosal surfaces are associated with activation of the complement, both by the immune complexmediated classical pathway as well as by direct alternative pathway activation by infected cells.70 Complement activation has been shown to enhance neutrophil-mediated cytotoxity of viral-infected cells.71 Activation of the complement by viralinfected cells could also lead to a non-lytic neutralization of the virus by enveloping the infected cells with antibody and complement-derived proteins, thus masking the surface glycoproteins and other structures needed for the attachment of the virus particle to potentially infectible cells.72 The complement system could also induce a direct lytic effect on certain virus-infected cells.73 It might be concluded that protection of the host against viral infection on the mucosal surfaces could not be achieved without a significant contribution by the complement system. Inflammatory reactions on mucosal surfaces Anaphylatoxin peptides in HBM may contribute to inflammatory reactions in the gastrointestinal tract by increasing vascular permeability and thereby increasing the leakage of serum antibodies and more complement, in addition to increased leucocyte migration, to effectively combat infectious agents.74,75 The AT are also known to mediate inflammatory responses through the regulation of the production of cytokines, such as IL-6.76 Animal experimental studies have shown that locally instilled AT are several fold more toxic on the pulmonary tissue than those generated in the circulation.77 This would suggest that even relatively low levels of AT present in the local mucosal secretions could play significant physiological roles. Sublytic levels of C5b-9 on tissue cells and leucocytes in mucosal secretions are also able to exert inflammatory functions, including the release of inflammatory mediators, such as thromboxane B2, other prostanoids, IL-1 and leukotrienes, as well as toxic oxygen radicals.78 Increased levels of AT peptide C3a in the tears of patients with conjunctivitis in the absence of immune complexes may be contributing to mast cell and basophil activation with the release of inflammatory mediators into tear secretions.49 The early appearance of leucocytosis and the clearance of bacteria during experimental bovine mastitis have been shown to precede any detectable increase in IL-1 and IL-6 activity, thereby suggesting a possible role for complement derived AT particularly C5a.79 This and other experimental evidence suggests a physiological role for complement-derived inflammatory mediators on mucosal surfaces. Immunomodulation of mucosal immunological reactions Anaphylatoxins C3a and C5a, as well as other C3 fragments, are known to modulate the immune responses of the host in the microenvironment of complement activation where they are present in relatively high concentrations.80 C5a is rapidly degraded by serum carboxypeptidase-N, which cleaves the functionally important carboxy-terminal arginine to form C5adesArg. C5adesArg enhances the primary polyclonal antibody responses of cultured peripheral blood leucocytes (PBL) in vitro to IgG1 derived Fc fragments and to sheep red blood cells (SRBC). These enhanced activities are abrogated by depletion of CD4 Th cells from the PBL, or by substitution of the T cells by a soluble T-cell replacing factor.80 However, C3a suppresses the primary polyclonal and antigen-specific antibody responses of both cultured human PBL and murine splenic cells. C3a and its synthetic octapeptide C3a(70–77) also inhibit the generation of Leucocyte Inhibitory Factor (LIF) by human lymphocytes in response to mitogens, with C3a being 10-fold more potent than the synthetic analogue. This inhibition is abrogated when T cells are substituted in the culture by soluble T-cell factors. C3b is known to enhance T cell-dependent humoral responses, although some of these effects are speculated to be attributable to factor H, which is released when C3b is bound to the cellular receptor.80,81 C3b may also act as a cofactor in the antigen-dependent activation of T cells. In this way, local C3 could facilitate recognition of foreign antigens by lymphocytes in breast milk and other mucosal secretions. The absence of potent serum enzymes, such as carboxypeptidases, in mucosal secretions, which normally control the level of circulating AT in the serum and restrict them to sites of inflammation, may indicate that these potent peptides play a significant physiological role on epithelial surfaces. Studies with nasal secretions have shown that carboxypeptidase-N enzyme can only be secreted from the plasma through the mucosa during inflammation of the respiratory tract, accompanied by increased vascular permeability.82 In the absence of inflammation, it is possible that even small quantities of AT peptides could exert significant physiological functions on the mucosal surfaces. Anti-allergic functions of mucosal complement Breast-feeding provides a prolonged benefit for infants in terms of protection against allergic diseases.83 C3a has recently been shown to be capable of inhibiting mast cell degranulation induced by the binding of antigens to cellbound IgE at nanomolar concentrations.84 C3a is also a potent immunosuppressor, which may modulate the production of IgE antibodies directed against food and other allergens.80 C3a may also be involved in the observed induction of a memory suppressor subset of lymphocytes, which regulates the production of IgE antibodies.85 The possibility of non-immune production of AT C3a in breast milk, for example by breast milk proteases and Roles of the mucosal complement system digestive enzymes, may lead to a high local concentration of this AT in the infant’s intestine. C3 is immunochemically present at a generally higher concentration in human milk compared with C5 (Table 1). Prostaglandins,86,87 in addition to AT, are also present in significant quantities in breast milk and both are powerful stimulants of mast cell secretion of histamine. Histamine acting via the H2 receptors is known to induce immunosuppression at high concentrations and immune stimulation at low concentrations. The histamine H2 receptor-bearing T lymphocytes function as suppressor cells for specific antibody production. Tolerance to orally presented antigen is attributed to induction of T-suppressor cells, which could be prevented by prior administration of H2 receptor antagonists or selective removal of histamine receptor-bearing cells in mice.88 This could be at least one of the mechanisms by which breast-fed infants are protected against undue sensitivity to environmental antigens leading to allergic reactions. Regulation of gut motility Anaphylatoxins are potent spasmogens and they could contribute to the increased gut motility in breast-fed infants. Other hormones and high levels of prostaglandins present in HBM have also been suggested to contribute to the regulation of an infant’s gut motility.89,90 The effect of C3a is apparently histamine-independent, while endogenous histamine may be involved in the spasmogenic effect of C5adesArg.91 Modulation of mucosal electrolyte secretions It has been shown that AT C5a modulates electrolyte secretion of the intestinal mucosa. This activity is mediated by histamine and cyclo-oxygenase products of arachidonic acid,92 and may account for changes in electrolyte and water composition associated with inflammation at mucosal sites.93 The AT C5a activity may also be responsible for the normal regulation of water and electrolyte contents in breast milk and other mucosal secretions. The recent discovery of C5a receptors on the intestinal epithelial mucosa provides further evidence for a potent physiological role of AT in an infant’s intestinal tract and other epithelial surfaces.94 Phlogistic effects of mucosal anaphylatoxins The complement-derived AT in human milk could act in consonance with some human milk cytokines to protect the mammary gland by attracting blood leucocytes to combat foreign antigens and pathogens invading the gland. In vitro and in vivo studies in cattle have shown that C5a, and other inflammatory mediators are able to induce significant accumulation of leucocytes, mainly neutrophils, into the milk.79 Other possible pointers to the phlogistic effects of human milk AT include the findings of characteristics similar to those produced by AT stimulation on human milk leucocytes, including enhanced expression of CD45RO.63 Many studies have shown that human milk cells are generally less reactive to chemotactic stimuli in vitro.95 A possible explanation for this could be the induction of a state of desensitization in these cells, after they have been activated and attracted into the milk.96 7 Possible apoptotic functions of mucosal complement A possible link between the complement system and apoptosis is provided by the observation that many of the membrane molecules that are downregulated on apoptotic cells belong to the complement regulatory molecule family, while the CR3 receptor is upregulated. Activation of the complement system has been shown to significantly enhance both the rate of apoptosis induction, as well as the recognition and phagocytosis of apoptotic cells by macrophages.97 Apoptotic cells are capable of activating the alternative pathway of complement and depositing iC3b on their cell membrane.98 Furthermore, one of the proteins in human milk, alpha lactalbumin, has been shown to be a specific inducer of apoptosis.99 It is of utmost importance to a newborn, that milk cells are disposed of in a way which prevents their lysis and the release of tissue-damaging enzymes and inflammatory mediators, especially in the early stages of lactation, where numbers of neutrophils approach that in the blood. Breast milk complement may enhance the process of apoptosis of milk neutrophils and favour their ingestion by milk macrophages after opsonization. Apoptosis also represents a mechanism for the removal of neutrophils during inflammation on mucosal surfaces, and thereby serves to limit the degree of underlying delicate tissue damage.100 Prospects in mucosal complement research Further research efforts are needed to answer many of the intriguing questions arising about the role and the significance of the complement system in mucosal immunity. Any apparent problems responsible for hindering research efforts on this subject need to be properly addressed by the development of appropriate standard laboratory techniques. The complement system should be regarded as a potentially significant source of contribution to humoral host resistance mechanisms, on epithelial surfaces, against infection and allergies. The limited experimental and clinical evidence points to the possible physiological significance of the complement system in mucosal immunity. Conclusion The complement system constitutes one of the many humoral host-resistant factors associated with ‘mucosal immunity’. These factors help to protect the body at its external interfaces with the environment. It is conceivable that several mechanisms could be used to safeguard the delicate internal millieu of the organism and to protect it from the onslaught of foreign and pathogenic environmental factors. The complement system could no doubt play a complementary role to other immunological and non-immunological protective mechanisms. In the present paper some of the identified and proposed possible roles for the complement system have been highlighted, to provoke further research and interest in this subject. Many studies show direct experimental evidence for localized synthesis and activation of various complement components at different mucosal sites. Several clinical studies have also suggested important local physiological roles for the complement system on mucosal surfaces. Some studies have 8 MO Ogundele demonstrated involvement of the mucosal complement system in bacterial opsonization and bacteriolysis, as well as in the production of chemotactic and inflammatory mediators. Indirect experimental evidence also suggests that the complement system is actively involved in cytotoxicity against viral-infected cells and modulation of mucosal immune responses. Other possible roles of the mucosal complement system requiring further study include modulation of allergic reactions, induction of apoptosis, physiological regulation of electrolyte secretions and gut motility. Acknowledgements This work and other reported personal experiments were performed at the Department of Immunology, Georg-August University Germany, during the tenure of a fellowship from the German Academic Exchange Service (DAAD). Financial support provided by the DAAD to the author during the preparation of this manuscript is hereby acknowledged. The kind support of Prof. O Götze and all the coscientists at the Immunology Department at Göttingen University is greatly appreciated. The kind assistance of Dr Robert Giesseler, who painstakingly reviewed the manuscript, is greatly appreciated. References 1 Mestecky J, McGhee JR, Michalek SM, Arnold RR, Crago SS, Babb JL. Concept of the local and common mucosal immune response. Adv. Exp. Med. Biol. 1978; 107: 185–92. 2 McGhee JR, Kiyono H, Michalek SM, Mestecky J. Enteric immunization reveals a T cell network for IgA responses and suggests that humans possess a common mucosal immune system. Antonie Van Leeuwenhoek 1987; 53: 537–43. 3 France GL, Marmer DJ, Steele RW. Breast-feeding and Salmonella infection. Am. J. Dis. Child. 1980; 134: 147–52. 4 Jason JM, Nieburg P, Marks JS. Mortality and infectious disease associated with infant-feeding practices in developing countries. Pediatrics 1984; 74: 702–27. 5 Haffejee IE, Moosa A. Windsor I. Circulating and breast-milk anti-rotaviral antibodies and neonatal rotavirus infection: a maternal-neonatal study. Ann. Trop. Paediatr. 1990; 10: 3–14. 6 Lodinova R, Jouja V. Antibody production by the mammary gland in mothers after artifical oral colonization of their infants with a non-pathogenic strain of E coli 083. Acta Paediatr. Scand. 1977; 66: 705–8. 7 Prentice A, Watkinson M, Prentice AM, Cole TJ, Whitehead RG. Breast-milk antimicrobial factors of rural Gambian mothers. 2. Influence of season and prevalence of infection. Acta Paediatr. Scand. 1984; 73: 803–9. 8 Prentice A, Prentice AM, Cole TJ, Paul AA, Whitehead RG. Breast-milk antimicrobial factors of rural Gambian mothers. I. Influence of stage of lactation and maternal plane of nutrition. Acta Paediatr. Scand. 1984; 73: 796–802. 9 Prentice A, Ewing G, Roberts SB, Lucas A, MacCarthy A, Jarjou LM, Whitehead RG. The nutritional role of breast-milk IgA and lactoferrin. Acta Paediatr. Scand. 1987; 76: 592–8. 10 Ebrahim GJ. Breast-milk immunology. J. Trop. Pediatr. 1995; 41: 2–4. 11 Goldman AS, Chheda S, Garofalo R, Schmalstieg FC. Cytokines in human milk: Properties and potential effects upon the mammary gland and the neonate. J. Mammary Gland Biol. Neoplasia 1996; 1: 251–8. 12 Avery VM, Gordon DL. Antibacterial properties of breast-milk: requirements for surface phagocytosis and chemiluminiscence. Eur. J. Clin. Microbiol. Infect. Dis. 1991; 10: 1034–9. 13 Thornburg W, Koldovsky O. Hormones in milk. A review. J. Pediatr. Gastroenterol. Nutr. 1987; 6: 172–96. 14 Barret JT. The Complement System. In: Barrett JT (ed.). Textbook of Immunology, 4th ed. London: CV Mosby Company, 1983; 170–200. 15 Chernyshov VP, Slukin II. [Osobennosti mestnogo immuniteta novorozhdennykh.] Characteristics of local immunity in newborn infants. Pediatriia 1989; 6: 20–4. 16 Loos M. The classical complement pathway: mechanism of activation of the first component by antigen-antibody complexes. Prog. Allergy 1982; 30: 135–92. 17 Götze O, Muller Eberhard HJ. The alternative pathway of complement activation. Adv. Immunol. 1976; 24: 1–35. 18 Goldman AS, Thorpe LW, Goldblum RM, Hanson LA. Antiinflammatory properties of human milk. Acta Paediatr. Scand. 1986; 75: 689–95. 19 Ogundele MO. Inhibitors of complement activity in human breast-milk: a proposed hypothesis of their physiological significance. Mediators Inflamm. 1999; 8: 69–75. 20 Goldman AS. The immune system of human milk: antimicrobial, anti-inflammatory and immunomodulating properties. Anti-inflammatory properties of human milk. Pediatr. Infec. Dis. J. 1993; 12: 664–71. 21 McClelland DB, McGrath J, Samson RR. Antimicrobial factors in human milk. Studies of concentration and transfer to the infant during the early stages of lactation. Acta Paediatr. Scand. 1978; 271: 1–20. 22 Cole FS, Schneeberger EE, Lichtenberg NA, Colten HR. Complement biosynthesis in human breast-milk macrophages and blood monocytes. Immunology 1982; 46: 429–41. 23 Jagadeesan V, Reddy V. C3 in human milk. Acta Paediatr. Scand. 1978; 67: 237–8. 24 Reddy V, Bhaskaram C, Raghuramulu N, Jagadeesan V. Antimicrobial factors in human milk. Acta Paediatr. Scand. 1977; 66: 229–32. 25 Kassim OO, Afolabi O, Ako-Nai KA et al. Immunoprotective factors in breast milk and sera of mother-infant pairs. Trop. Geogr. Med. 1986; 38: 362–6. 26 Ballow M, Fang F, Good RA, Day NK. Developmental aspects of complement components in the newborn. The presence of complement components and C3 proactivator (properdin factor B) in human colostrum. Clin. Exp. Immunol. 1974; 18: 257–66. 27 Yonemasu K, Kitajima H, Tanabe S, Ochi T, Shinkai H. Effect of age on C1q and C3 levels in human serum and their presence in colostrum. Immunology 1978; 35: 523–30. 28 Nakajima S, Baba AS, Tamura N. Complement system in Human Colostrum. Int. Arch. Allergy Appl. Immunol. 1977; 54: 428–33. 29 Oppermann M, Höpkin U, Götze O. Assessment of complement activation in vivo. Immunopharmacology 1992; 24: 119–134. 30 Thomas L. Labor und Diagnose (Laboratory and Diagnosis), 4th edn Marburg, Germany: Die medizinische Verlagsgesellschaft, 1992. 31 Tanzer F, Sunel S. Calcium, magnesium and phosphorus concentrations in human milk and in sera of nursing mothers and their infants during 26 weeks of lactation. Indian Pediatr. 1991; 28: 391–400. 32 Oppermann M, Baumgarten H, Brandt E, Gottsleben W, Kurts C, Götze O. Quantification of components of the alternative pathway of complement (APC) by enzyme-linked immunosorbent assays. J. Immunol. Methods 1990; 133: 181–90. Roles of the mucosal complement system 33 Feeley RM, Eitenmiller RR, Jones Jr JB, Barnhart H. Calcium, phosphorus and magnesium contents of human milk during early lactation. J. Pediatr. Gastroenterol. Nutr. 1983; 2: 262–7. 34 Mondino BJ, Zaidman GW. Hemolytic complement in tears. Ophthalmic Res. 1983; 15: 208–11. 35 Reiter B, Brock JH. Inhibition of Escherichia coli by bovine colostrum and post-colostral milk. I. Complement-mediated bactericidal activity of antibodies to a serum susceptible strain of E. coli of the serotype 0111. Immunology 1975; 28: 71–82. 36 Poutrel B, Caffin JP. A sensitive microassay for the determination of hemolytic complement activity in bovine milk. Vet. Immunol. Immunopathol. 1983; 5: 177–84. 37 Minta JO, Jezyk PD, Lepow ICH. Distribution and levels of properdin in human body fluids. Clin. Immunol. Immunopathol. 1976; 5: 84–90. 38 Alper CA, Johnson AM, Birtch AG, Moore FD. Human C3: evidence for the liver as the primary site of synthesis. Science 1969; 163: 286–8. 39 Colten HR, Gordon JM, Rapp HJ, Borsos T. Synthesis of the first component of guinea pig complement by columnar epithelial cells of the small intestine. J. Immunol. 1968; 100: 788–92. 40 Andrews PA, Finn JE, Lloyd CM, Zhou W, Mathieson PW, Sacks SH. Expression and tissue localization of donor-specific complement C3 synthesized in human renal allografts. Eur. J. Immunol. 1995; 25: 1087–93. 41 Lai-A-Fat RF, McClelland DB, van-Furth R. In vitro synthesis of immunoglobulins, secretory component, complement and lysozyme by human gastrointestinal tissues. I. Normal tissues. Clin. Exp. Immunol. 1976; 23: 9–19. 42 Passwell J, Schreiner GF, Nonaka M, Beuscher HU, Colten HR. Local extrahepatic expression of complement genes C3, factor B, C2 and C4 is increased in murine lupus nephritis. J. Clin. Invest. 1988; 82: 1676–84. 43 Ahrenstedt O, Knutson L, Nilsson B, Nilsson-Ekdahl K, Odlind B, Hallgren R. Enhanced local production of complement components in the small intestines of patients with Crohn’s disease. N. Engl. J. Med. 1990; 322: 1345–9. 44 Andoh A, Fujiyama Y, Bamba T, Hosoda S. Differential cytokine regulation of complement C3, C4 and factor B synthesis in human intestinal epithelial cell line, Caco-2. J. Immunol. 1993; 151: 4239–47. 45 Russell MW, Mansa B. Complement-fixing properties of human IgA antibodies. Alternative pathway complement activation by plastic-bound, but not specific antigen-bound, IgA. Scand. J. Immunol. 1989; 30: 175–83. 46 Russell MW, Reinholdt J, Kilian M. Anti-inflammatory activity of human IgA antibodies and their Fab alpha fragments: inhibition of IgG-mediated complement activation. Eur. J. Immunol. 1989; 19: 2243–9. 47 Rainard P, Poutrel B. Deposition of complement components on Streptococcus agalactiae in bovine milk in the absence of inflammation. Infect. Immun. 1995; 63: 3422–7. 48 Camussi G, Stratta P, Mazzucco G, Gaido M, Tetta C, Castello R, Rotunno M, Vercellone A. In vivo localization of C3 on the brush border of proximal tubules of kidneys from nephrotic patients. Clin. Nephrol. 1985; 23: 134–41. 49 Ballow M, Donshik PC, Mendelson L. Complement proteins and C3 anaphylatoxin in the tears of patients with conjunctivitis. J. Allergy Clin. Immunol. 1985; 76: 473–6. 50 Imanishi J, Takahashi F, Inatomi A, Tagami H, Yoshikawa T, Kondo M. Complement levels in human tears. Jpn. J. Ophthalmol. 1982; 26: 229–33. 9 51 Grieve PA, Kitchen BJ. Proteolysis in milk: the significance of proteinases originating from milk leucocytes and a comparison of the action of leucocyte, bacterial and natural milk proteinases on casein. J. Dairy Res. 1985; 52: 101–12. 52 Linberg T, Ohlsson K, Westrom B. Protease inhibitors and their relation to protease activity in human milk. Pediatr. Res. 1982; 16: 479–83. 53 Kijlstra A, Jeurissen SH, Koning KM. Complement inhibitors in tears of different species. Exp. Eye Res. 1984; 38: 57–62. 54 Zalman LS, Brothers MA, Müller-Eberhard HJ. Isolation of homologous restriction factor from human urine. Immunochemical properties and biologic activity. J. Immunol. 1989; 143: 1943–7. 55 Schroten H, Hanisch FG, Plogmann R et al. Inhibition of adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of the protective function of mucins in the nonimmunoglobulin fraction. Infect. Immun. 1992; 60: 2893–9. 56 Hakulinen J, Meri S. Shedding and enrichment of the glycolipid anchored complement lysis inhibitor protectin (CD59) into milk fat globules. Immunology 1995; 85: 495–501. 57 Lass JH, Walter EI, Burris TE et al. Expression of two molecular forms of the complement decay accelerating factor in the eye and lacrimal gland. Invest. Ophthalmol. Vis. Sci. 1990; 31: 1136–48. 58 Medof ME, Walter EI, Rutgers JL, Knowles DM, Nussenzweig V. Identification of the complement decay accelerating factor (DAF) on epithelium and glandular cells and in body fluids. J. Exp. Med. 1987; 165: 848–64. 59 Sack RA, Underwood A, Tan KO, Morris C. Vitronectin in human tears—protection against closed eye induced inflammatory damage. Adv. Exp. Med. Biol. 1994; 350: 345–9. 60 Prentice A, Prentice AM, Lamb WH. Mastitis in rural Gambian mothers and the protection of the breast milk by antimicrobial factors. Trans. R. Soc. Trop. Med. Hyg. 1985; 79: 90–5. 61 Ogundele MO. Complement mediated bactericidal activity of human milk to a serum susceptible strain of E. coli 0111. J. Appl. Microbiol. 1999; 87: 689–96. 62 Frank MM, Fries LF. The role of complement in inflammation and phagocytosis. Immunol. Today 1991; 12: 322–6. 63 Werfel T, Sonntag G, Weber MH, Götze O. Rapid increases in the membrane expression of neutral endopeptidase (CD10), aminopeptidase N (CD13), tyrosine phosphatase (CD45) and Fc gamma RIII (CD16) upon stimulation of human peripheral leucocytes with human C5a. J. Immunol. 1991; 147: 3909–14. 64 Wardlaw AC. The complement-dependent bacteriolytic activity of normal human serum. I. The effect of pH and ionic strength and the role of lysozyme. J. Exp. Med. 1962; 115: 1231–49. 65 Kubens BS, Opferkuch W. Defense against bacterial infections. In: Till GO, Rother K (eds) The Complement System. Berlin: Springer Verlag, 1988; 469–87. 66 Rainard P, Poutrel B, Caffin JP. Assessment of hemolytic and bactericidal complement activities in normal and mastitic bovine milk. J. Dairy Sci. 1984; 67: 614–9. 67 Knoop U, Schutt Gerowitt H, Matheis G. Bacterial growth in breast milk under various storage conditions (Bakterienwachstum bei unterschiedlischer Lagerung von Muttermilch.) Monatschr. Kinderheilkd, 1985; 133: 483–6. 68 Yoshioka H, Iseki K, Fujita K. Development and differences of intestinal flora in the neonatal period in breast fed and bottle fed infants. Pediatrics 1983; 72: 317–21. 69 Van-de-Perre P, Simonon A, Hitiamana DG et al. Infective and anti-infective properties of breast-milk from HIV-1 infected women Lancet 1993; 341: 914–8. 10 MO Ogundele 70 Kaul TN, Welliver RC, Ogra PL. Appearance of complement components and immunoglobulins on nasopharyngeal epithelial cells following naturally acquired infection with respiratory syncytial virus. J. Med. Virol. 1982; 9: 149–58. 71 Kaul TN, Faden H, Baker R, Ogra PL. Virus induced complement activation and neutrophil mediated cytotoxicity against respiratory syncytial virus (RSV). Clin. Exp. Immunol. 1984; 56: 501–8. 72 Cooper NR, Nemerow GR. Complement, viruses and virus infected cells. Springer Semin. Immunopathol. 1983; 6: 327–47. 73 Bartholomew RM, Esser AF, Muller Eberhard HJ. Lysis of oncornaviruses by human serum. Isolation of the viral complement (C1) receptor and identification as p15E. J. Exp. Med. 1978; 147: 844–53. 74 Jose PJ, Forrest MJ, Williams TJ. Human C5a des Arg increases vascular permeability. J. Immunol. 1981; 127: 2376–80. 75 Luo HY, Wead WB, Yang S, Wilson MA, Harris PD. Nitric oxide mediates C5a-induced vasodilation in the small intestine. Microcirculation 1995; 2: 53–61. 76 Höpken U, Mohn M, Strüber A et al. Inhibition of interleukin-6 synthesis in an animal model of septic shock by anti-C5a mononuclear antibodies. Eur. J. Immunol. 1996; 26: 1103–9. 77 Stimler NP, Hugli TE, Bloor CM. Pulmonary injury induced by C3a and C5a anaphylatoxins. Am. J. Pathol. 1980; 100: 327–38. 78 Hänsch GM, Seitz M, Betz M. Effect of the late complement components C5b-9 on human monocytes: release of prostanoids, oxygen radicals and of a factor inducing cell proliferation. Int. Arch. Allergy Appl. Immunol. 1987; 82: 317–20. 79 Schuster DE, Kehrli Jr ME, Stevens MG. Cytokine production during endotoxin-induced mastitis in lactating dairy cows. Am. J. Vet. Res. 1993; 54: 80–5. 80 Weigle WO, Morgan EL, Goodman MG, Chenoweth DE, Hugli TE. Modulation of the immune response by anaphylatoxin in the microenvironment of the interacting cell. Fed. Proc. 1982; 41: 3099–103. 81 Arvieux J, Yssel H, Colomb MG. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones. Immunology 1988; 65: 229–35. 82 Levin Y, Skidgel RA, Erdos EG. Isolation and characterization of the subunits of human plasma carboxypeptidase N (kininase i). Proc. Natl Acad. Sci. USA 1982; 79: 4618–22. 83 Saarinen UM, Kajosaari M. Breastfeeding as prophylaxis against atopic disease: Prospective follow-up study until 17 years old. Lancet 1995; 346: 1065–9. 84 Erdel A, Andreev S, Pecht I. Complement peptide C3a inhibits IgE-mediated triggering of rat mucosal mast cells. Int. Immunol. 1995; 7: 1433–9. 85 Schwenk RJ, Kudo K, Miyamoto M, Sehon AH. Regulation of the murine IgE antibody response. I. Characterization of 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 suppressor cells regulating both persistent and transient responses and their sensitivity to low does of X-irradiation. J. Immunol. 1979; 123: 2791–8. Bedrick AD, Britton JR, Johnson S, Koldovsky O. Prostaglandin stability in human milk and infant gastric fluid. Biol. Neonate 1989; 56: 192–7. Lucas A, Mitchell MD. Prostaglandins in human milk. Arch. Dis. Child. 1980; 55: 950–2. Schnitzler S, Eckert R, Volk D, Gronow R. Histamin und Immunoreaktionen. Histaminrezeptor-tragende Lymphozyten (Histamine receptor-bearing lymphocytes). Allerg. Immunol. Leipz. 1982; 28: 219–35. Sanderson SD, Kirnarsky L, Sherman SA, Ember JA, Finch AM, Taylor SM. Decapeptide agonists of human C5a: the relationship between conformation and spasmogenic and platelet aggregatory activities. J. Med. Chem. 1994; 37: 3171–80. Cavell B. Gastric emptying in infants fed human milk or infant formula. Acta Paediatr. Scand. 1981; 70: 639–41. Sorgenfrei J, Damerau B, Vogt W. Role of histamine in the spasmogenic effect of the complement peptides C3a and C5adesArg (classical anaphylatoxin). Agents Actions 1982; 12: 118–21. Kachur JF, Won-Kim S, Anglin C, Gaginella TS. Eicosandoids and histamine mediate C5a-induced electrolyte secretion in guinea pig ileal mucosa. Inflammation 1995; 19: 717–25. Racusen LC, Binder HJ. Effect of prostaglandin on ion transport across isolated colonic mucosa. Dig. Dis. Sci. 1980; 25: 900–4. Wetsel RA. Expression of the complement C5a anaphylatoxin receptor (C5aR) on non-myeloid cells. Immunol. Lett. 1995; 44: 183–7. Thorpe LW, Rudloff HE, Powell LC, Goldman AS. Decreased response of human milk leucocytes to chemoattractant peptides. Pediatr. Res. 1986; 20: 373–7. Meuer S, Zanker, B. Hadding U, Bitter-Suermann D. Low zone desensitization: a stimulus-specific control mechanism of cell response. Investigations on anaphylatoxin-induced platelet secretion. J. Exp. Med. 1982; 155: 698–710. Mevorach D, Mascarenhas JO, Gershov D, Elkon KB. Complement-dependent clearance of apoptotic cells by human macrophages. J. Exp. Med. 1998; 188: 2313–20. Matsui H, Tsuji S, Nishimura H, Nagasawa S. Activation of the alternative pathway of complement by apoptotic Jurkat cells. FEBS Lett. 1994; 351: 419–22. Hakansson A, Zhivotovsky B, Orrenius S, Sabharwal H, Svanborg C. Apoptosis induced by a human milk protein. Proc. Natl Acad. Sci. USA 1995; 92: 8064–8. Meagher LC, Savill JS, Baker A, Fuller RW, Haslett C. Phagocytosis of apoptotic neutrophils does not induce macrophage release of thromboxane B2. J. Leukoc. Biol. 1992; 52: 269–73.