Download Role and significance of the complement system in mucosal

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.
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