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Department of Immunology/Microbiology, Rush Medical College, Chicago, IL 60612
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MBL is an acute phase protein (18, 19) that is thought to be an important constituent of the innate immune system (14, 17, 20, 21). It plays a crucial role in host defense against certain pathogens that contain ligands for MBL on their surfaces. It can function as an opsonin. Native and recombinant human MBL are able to directly bind to wild-type virulent Salmonella montevideo expressing a mannose-rich O-polysaccharide; this results in the attachment, uptake, and killing of MBL-coated bacteria by phagocytes in the absence of serum (22). MBL deficiency, which is linked to three allelic mutations in codons 52, 54, and 57 of the first exon of the MBL gene (23, 24, 25), is associated with a common opsonic defect that results in recurrent or persistent infection early in life (26, 27). It recently was shown that homozygous carriers of these variant MBL alleles are at increased risk of HIV infection (28).
MBL and C1q have a similar ultrastructural organization, even though they do not share amino acid sequence homology (14, 15, 16, 17, 29, 30). MBL is able to activate the complement system via the classical pathway (31, 32, 33, 34, 35, 36) and to bind to the surface receptor for C1q (30, 37, 38). However, the newly discovered serine proteases MASP-1 (35, 39) and MASP-2 (40), rather than the homologous C1r and C1s, were shown to associate with MBL and to be required for MBL to activate C4, C2, and hence the rest of the complement cascade; this series of interactions has been termed the lectin pathway of complement activation. However, MBL also has been reported to trigger the complement system through the alternative pathway. Thus, it was reported that MBL-mediated deposition of C3 on mannose-rich Salmonella occurred via the alternative pathway without classical pathway involvement (41), and that MASP is able to cleave C3 directly with subsequent activation of the alternative pathway (42).
In the present paper we sought to further clarify the mechanism by which the lectin pathway leads to complement activation in human serum. We report here that the alternative pathway as well as C4 and C2 are required for complement activation by the lectin pathway, and we present a new assay for quantifying lectin pathway activity in normal human serum.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gelatin-veronal-buffered saline consisting of 5 mM veronal (pH 7.4), 0.145 M NaCl, and 0.1% gelatin (GVB); GVB containing 2 mM CaCl2 and 0.5 mM MgCl2 (GVB2+); GVB containing 0.5 mM MgCl2; GVB containing 0.5 mM CaCl2; a mixture of equal parts of GVB2+ and 5% glucose2+ to make the ionic strength (µ/2) equal to 0.075 NaCl (GGVB2+); GVB containing 0.5 mM MgCl2, 100 mM ethylene glycoltetraacetic acid, and glucose to make the ionic strength equal to 0.075 M NaCl (Mg-EGTA); and GVB containing 10 mM EDTA (EDTA-GVB) were prepared as previously described (31, 43).
Reagents
Saccharomyces cerevisiae mannan was purchased from Sigma Chemical Co. (St. Louis, MO). Anti-MBL mAb was purchased from Statens Serum Institute (Copenhagen, Denmark). R-phycoerythrin-conjugated streptavidin for use in flow cytometry was purchased from Dako Co. (Glostrup, Denmark).
Sera and complement components
Aliquots of normal human serum (NHS) collected from healthy, normal donors and from patients congenitally lacking individual complement components were stored at -70°C. Every complement-deficient serum used was shown to have <2% normal hemolytic activity, with normal levels restored upon addition of small amounts of the purified deficient component. Serum deficient in both C1q and factor D was prepared by column chromatography on Bio-Rex 70 (Bio-Rad, Hercules, CA) (44). Removal of C1q was ascertained by lysis of Ab-sensitized sheep erythrocytes in the presence and absence of added C1q; depletion of factor D was documented by lysis of rabbit erythrocytes in Mg-EGTA in the presence and absence of added factor D. Purified human C1 and C1q and guinea pig C2 were prepared as previously described (44, 45). Purified human C4, C2, and factors D and B as well as factor B-depleted human serum were purchased from Calbiochem (La Jolla, CA).
ELISA for MBL
MBL concentrations were assayed using a minor modification of previously described sandwich ELISA methodology (27). Briefly, microtiter plates were coated with monoclonal anti-MBL (3 µg/ml, 100 µl/well) in coating buffer (0.03 M Na2CO3 and 0.02 M NaHCO3, pH 9.6) and incubated overnight at 4°C. The microtiter plates were washed three times with veronal (5 mM)-buffered saline (pH 7.4) containing 0.05% Tween 20 (VBS-T) and blocked for 2 h at room temperature by adding 200 µl VBS containing 1% BSA to each well. Samples and MBL standards were loaded into the wells in duplicate, incubated at room temperature for 1 h, and washed as before. Biotinylated monoclonal anti-MBL diluted in VBS (100 µl, 1 µg/ml) was added to each well, followed by incubation at room temperature for 1 h. The plates were washed, streptavidin-horseradish peroxidase conjugate (1/1000 in VBS, 100 µl/well) was added, incubation was continued at room temperature for 1 h, and after additional washes, 100 µl substrate was added. Incubation was continued for 30 min at room temperature, the reaction was stopped by addition of 2 N HCl (100 µl/well), and the absorbance at 450 nm was read on a microplate reader.
Mannan-binding lectin
MBL was prepared by sequential affinity column chromatography by minor modification of the method of Kawasaki et al. (8). Briefly, human serum obtained by recalcification of pooled human citrated plasma of known MBL concentration was brought to 20 mM CaCl2 and allowed to clot for 1 h at 37°C and overnight at 4°C. After removal of the clot, the serum was dialyzed extensively against starting buffer containing 50 mM Tris-HCl, 1 M NaCl, 20 mM CaCl2, and 0.05% (w/v) NaN3 (pH 7.8). After centrifugation at 10,000 x g for 10 min, the supernatant was applied to a mannan-Sepharose 4B column (100 ml) that had been equilibrated with starting buffer. The column was washed with starting buffer, and the bound proteins were eluted with a buffer containing 50 mM Tris-HCl, 1 M NaCl, and 20 mM EDTA, pH 7.8. The eluate was brought to 50 mM CaCl2 and reapplied to a second, smaller (25 ml) but otherwise identical, mannan-Sepharose 4B column, and the bound proteins were eluted in the same manner as in the first column.
Preparation of hemolytic intermediate cells
Ab-sensitized sheep erythrocytes (EA) (45), and erythrocytes (E) coated with mannan (E-M) and sensitized with MBL (E-M-MBL) were prepared as previously described (31, 46). Briefly, 0.5 ml of sheep E (1 x 109 cells/ml) were mixed with 0.5 ml of CrCl3 solution (0.5 mg/ml), 0.5 ml of mannan solution (200 µg/ml) was added, and the mixture was incubated with occasional shaking for 5 min at 25°C. The reaction was stopped by adding 1.5 ml of ice-cold GVB2+, and the E-M were washed three times and resuspended to a final concentration of 1 x 109 cells/ml in GVB2+. An aliquot (0.1 ml) was added to 0.4 ml of MBL (1–4000 ng) in GVB2+, incubated with gentle shaking for 15 min at room temperature, washed, and resuspended to 1 x 108 cells/ml in Mg-EGTA.
C4-coated indicator cells (EAC4) were prepared as described by Borsos and Rapp (47). Briefly, EA were washed three times and resuspended to 5 x 108 cells/ml in GGVB2+ prewarmed to 30°C. An equal volume of purified human C1 in GGVB2+ was added slowly with continuous shaking, and after 15-min incubation at 30°C, the mixture was washed, resuspended to the original volume in ice-cold GGVB2+, and cooled to 0°C in an ice-water bath. An equal volume of NHS diluted 1/4 in ice-cold EDTA-GVB was added and incubated with shaking for 15 min at 0°C. The mixture was washed three times and incubated at 37°C in EDTA-GVB to remove C1 and decay any C2 incorporated into EAC42, and the resulting EAC4 suspension was washed and resuspended to the original volume in GGVB2+. The Tmax was determined after addition of purified C1, as previously described (48), to establish the optimal NHS concentration to use in preparation of EAC4 and to quantitate the amount of C4 on the cells. EAC4-M were prepared by coating EAC4 with M as described above. EAC43 also were prepared as previously described (45).
Hemolytic assays
The CH50 and AH50 were determined as previously described (43, 45). Lectin pathway activity (LH50) was assayed by lysis of E-M-MBL in Mg-EGTA as follows. E-M-MBL (100 µl containing 1 x 108 cells/ml in Mg-EGTA) were incubated with 100 µl of test human serum in Mg-EGTA at 37°C for 1 h and centrifuged, and the OD414 of the supernatant was determined. The percent lysis and the amount of serum generating 50% lysis were determined in the usual way (43, 45); units are expressed as the dilution of serum added in 0.1 ml yielding 50% hemolysis.
Flow cytometry assay of MBL on sheep E
E-M-MBL (100 µl containing 1 x 108 cells/ml) were mixed with 100 µl of 1/50 diluted biotinylated anti-human MBL and incubated at 4°C for 1 h. After washing three times, 100 µl of 1/5 diluted R-phycoerythrin-conjugated streptavidin solution was added, and incubation proceeded for 1 h at 4°C. The washed cells were resuspended to 1 ml and assayed for MBL using an Ortho Cytoron flow cytometer (Ortho Diagnostic Systems, Raritan, NJ) to quantitate orange fluorescence. The fluorescence of cells is given as the mean channel intensity of 1 x 104 cells as measured using logarithmic amplification, except as noted. In this configuration, a 22-channel increase represents an approximate doubling of fluorescence.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In experiments on complement activation initiated by MBL, we
observed that E-M sensitized with MBL were lysed when added to NHS in
the presence of Mg-EGTA. Lysis occurred only with E-M sensitized with
MBL; neither E-M in the absence of MBL nor Ab-sensitized E was lysed
under these conditions. Lysis was directly proportional to the amount
of MBL and complement offered; 1000 ng of MBL/108 E-M
was optimal (Fig. 1
). We determined the
serum dilution required for 50% lysis of MBL-sensitized E-M in Mg-EGTA
(LH50 units) in NHS from 12 healthy individuals. As shown
in Figure 2, the mean LH50 of
E-M-MBL was 9 ± 8 U/ml (range, 1–25 U/ml). This surprising
occurrence of MBL-mediated hemolysis in the absence of calcium
contrasts with the calcium requirement for C1q-mediated hemolysis in
the classical complement pathway. However, these titers are
significantly less than the titers of 315 CH50 U/ml for
lysis of EA (SD = 72; range, 160–400 U/ml) and 91
AH50 U/ml (SD = 24; range, 59–140 U/ml) for lysis of
EAC43 when the classical and alternative pathways, respectively, were
assayed in the same serum samples using identical reaction volumes and
numbers of indicator cells.
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To further characterize and enhance this system for study of the
lectin pathway in human serum, we tested the ability of EAC4-M
sensitized with MBL for lysis in human serum diluted in Mg-EGTA. These
cells were significantly more susceptible to lysis in Mg-EGTA than were
E-M. Again, lysis was proportional to the amount of MBL used to
sensitize EAC4-M (2000 ng MBL/108 cells was optimal), and
significantly smaller amounts of human serum were required for 50%
hemolysis (Fig. 3). The mean MBL-mediated
hemolysis of EAC4-M-MBL in Mg-EGTA was 64 ± 15
LH50 U/ml (range, 50–100 U/ml), more than sevenfold
greater than lysis of E-M-MBL (Fig. 2
). The dependence of C4
enhancement on MBL-mediated hemolysis was further characterized. Lysis
was greater as greater amounts of C4 were used for preparation of EAC4
(Fig. 4); the Tmax of
optimally reactive cells prepared with 1/4 diluted human serum was 5
min. These results indicated that MBL-mediated hemolysis was enhanced
by C4, and that EAC4-M could serve as a valuable reagent for further
study of lectin pathway activity.
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We next examined the role of C2 in MBL-mediated hemolysis of
EAC4-M, using sera from five different individuals with inborn
deficiency of C2. As shown in Figure 5,
virtually no lysis occurred upon incubation of EAC4-M-MBL in any of
these sera, while the addition of purified human C2 (200 U/ml) restored
lysis to normal or near-normal levels (62 ± 23
LH50 U/ml). These results emphasize that
complement-dependent hemolysis of E-M-MBL in Mg-EGTA is indeed
complement dependent and proceeds through the classical pathway.
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Factor D and C1q were depleted from NHS by affinity column
chromatography on Bio-Rex 70, and this depleted serum was tested for
the capacity to mediate hemolysis via the lectin pathway. No lysis
occurred when EAC4-M-MBL were incubated in the depleted serum, even
when it was reconstituted with C1q (Fig. 6A). However, normal
lysis was restored upon reconstitution with factor D. Restoration of
lysis was directly dependent upon the amounts of factor D added; 0.25
µg was optimal (Fig. 6B). To further test whether
the alternative pathway is required for complement-dependent lysis via
the lectin pathway, experiments were performed with factor B-depleted
human serum. As shown in Figure 7, only
minimal lysis occurred when EAC4-M-MBL were incubated in factor
B-depleted serum, while normal lysis restored upon reconstitution with
300 µg/ml purified human factor B. The loss of lysis was incomplete
because of incomplete depletion of factor B, as shown when mixing this
depleted serum with EAC43 to assay for the presence of alternative
pathway activity. Collectively, these results indicate that
MBL-mediated hemolysis requires the alternative as well as the
classical pathway.
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We confirmed that the MBL-initiated lytic activity was indeed
mediated via the lectin pathway by performing hemolytic assays using
several different control cells and sera. Thus, EAC14 in the presence
or absence of MBL were not lysed in NHS-Mg-EGTA, indicating that even
when Ig and C1 were known to be present on the indicator cells, they
could not lead to lysis under the conditions of the assay used in
Mg-EGTA. Similarly, lysis of EAC4-M-MBL proceeded comparably in
C1q-depleted NHS and in agammaglobulinemic sera in Mg-EGTA as it had in
NHS, further excluding a requirement for C1 and Ig (Figs. 6A
and 8). Further, E-M incubated in
agammaglobulinemic serum diluted in calcium-containing buffer (i.e., in
GGVB2+) lysed only when the cells were presensitized with
MBL, and then to a similar degree (average titer, 8.3 CH50
U/ml) as lysis of E-M-MBL when these same sera were assayed in Mg-EGTA
(average titer, 7.5 CH50 U/ml). These results strengthen
the conclusion that the lytic activity is mediated via the lectin
pathway, and that the degree of lysis observed in the assay systems in
Mg-EGTA is reflective of the degree of lysis in calcium-containing
buffers.
Calcium-independent retention of MBL on E-M
Because E-M-MBL were lysed via the lectin pathway in NHS-Mg-EGTA,
we wondered whether MBL was retained on the E-M surface upon exposure
to and washes with Mg-EGTA. We investigated the effect of divalent
cation on binding and retention of MBL on E-M by flow cytometry. It can
be seen in Figure 9 that calcium was
required for binding of MBL on E-M, but, surprisingly, MBL was retained
on E-M independent of calcium, even in Mg-EGTA, EGTA, and EDTA. This
retention of MBL was selective and seemed to involve a ligand present
on the erythrocyte surface distinct from mannan, because comparable
calcium-independent retention was not observed on mannan immobilized on
polystyrene plates or beads (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We were surprised by the lysis of E-M-MBL in NHS in the presence of a calcium chelator, since MBL is a C-type lectin, and indeed, most procedures for purification of MBL involve elution by calcium chelation from mannose- or mannan-conjugated Sepharose. We therefore carefully investigated retention as well as binding of MBL to E-M and observed that in contrast to binding, short term retention of MBL occurred not only in the absence of calcium but also in the presence of both EGTA and EDTA. This calcium-independent retention of MBL on E-M is not seen in MBL reactions with mannan coated on polystyrene plates or conjugated to Sepharose beads, suggesting that it involves binding to a still undefined ligand present on the erythrocyte surface. This is consistent with an earlier study postulating an erythrocyte binding site that becomes available on MBL only consequent to its reactivity with mannan (31). Hemolysis of E-M-MBL in NHS-Mg-EGTA implies that calcium is not required for the association of the MASPs with MBL, consistent with the observation that activated MASP associates with MBL independent of calcium (49) and in striking contrast to an absolute calcium requirement for the association of C1q with C1r and C1s; it is not yet clear whether magnesium or another divalent cation other than calcium is required. We do not know whether MASP activity is provided by the MBL source (and hence absorbed to E-M before addition of NHS-Mg-EGTA), by NHS-Mg-EGTA, or by both, but in each of these possibilities, MASP persistence on E-M would be dependent on persistence on the erythrocyte of the MBL with which it is associated. In any case, calcium-independent retention of MBL on E-M is a fundamental characteristic of the lectin pathway activity described in this report.
It is striking that the classical pathway is necessary, but not sufficient, for hemolysis initiated by MBL-MASP complexes; factors D and B, and hence the alternative pathway, also are required for this lytic activity to occur. It is not yet clear why the alternative pathway is required. Perhaps inefficient C4 and C2 consumption, binding, and/or convertase assembly by E-MBL-MASP; decreased consumption and binding of C3 and/or C5; or the effect of control proteins is responsible. It cannot be attributable to decreased C4 binding alone, since even when the indicator cells were maximally presensitized with C4, alternative pathway amplification still was needed for lysis to occur. Further, an increase in C4 binding via the lectin pathway to levels well in excess of the minimum associated with Ab-initiated hemolysis via the classical pathway failed to result in hemolysis in absence of alternative pathway amplification (our unpublished observations). In any case, the alternative pathway requirement is consistent with a previous report that MBL activates the complement system via the alternative pathway (36). This latter report, which involved C3 deposition on mannose-rich Salmonella in NHS-Mg-EGTA, stated that this occurred via the alternative pathway alone, since it proceeded in a C2-deficient serum, whereas in the present report MBL-initiated lysis did not occur in any of five C2-deficient sera until C2 was reconstituted. Perhaps this difference is attributable to the different assay systems used, with some (if not maximal) C3 deposition occurring particularly on appropriate bacteria in the absence of C2. It also is not yet clear whether the recently reported direct C3 cleavage by MBL-MASP complexes (42) is involved in lectin pathway-initiated hemolysis, perhaps via the previously described enhancement of terminal attack complex activity on cells presensitized with C3b (50, 51). Whatever the mechanism involved, clearly both the classical and the alternative pathways are required for the MBL-initiated hemolysis described herein.
Hemolysis in NHS-Mg-EGTA of E-M presensitized with MBL in the presence of calcium proved to be a sensitive, quantitative, and convenient assay for lectin pathway activity, generating titers significantly greater than those previously described. Activity was greater still when EAC4 coated with mannan and sensitized with MBL were used, and further optimization of both systems may be possible. The basis for the enhanced activity by cells precoated with C4 is not yet clear, but may derive from relatively inefficient C4 binding via the lectin pathway, improvement of C3/C5 convertase formation, counteracting the effects of a lectin pathway inhibitory mechanism, and/or favoring of alternative pathway recruitment. Whatever the mechanism, we anticipate that assays in Mg-EGTA involving C4-coated E-M presensitized with MBL will be of use in further studies of the lectin pathway in the same manner that the EAC43 cell proved to be invaluable in analysis of the alternative pathway (52).
A schematic diagram summarizing the interactions of the lectin pathway
as indicated by the present report compared with those of the classical
and alternative pathways is shown in Figure 10. It remains to be established
whether this interpretation also applies to lectin pathway activation
by indicator particles other than E-M, such as bacteria, species of
serum other than human, and complement-dependent interactions other
than hemolysis. By its interactions with mannose moieties and other
ligands (14, 17, 21), MBL thus can initiate complement activation and
lead to complement-dependent phagocytosis and lysis in a distinctive
manner even in the absence of Ab. Studies of patients with deficiency
of MBL have indicated that the lack of this pathway is associated with
the loss of normal host defense (23, 24). Further investigation of this
newly appreciated mode of complement activation should yield insight
into the role of complement and acute phase proteins in innate
immunity, inflammation and host defense.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Henry Gewurz, Department of Immunology/Microbiology, Rush Medical College, Chicago, IL 60612.
3 Abbreviations used in this paper: MBL, mannan-binding lectin; MASP-1 and MASP-2, mannan-binding lectin-associated serine proteases structurally homologous to C1r and C1s, respectively; GVB, gelatin-veronal-buffered saline; GVB2+, gelatin-veronal-buffered saline containing 2 mM CaCl2 and 0.5 mM MgCl2; GGVB, a mixture containing equal parts of 5% glucose and gelatin-veronal-buffered saline; GGVB2+, GGVB containing 2 mM CaCl2 and 0.5 mM MgCl2; EDTA-GVB, gelatin-veronal-buffered saline containing 10 mM EDTA; NHS, normal human serum; VBS-T, veronal (5 mM)-buffered saline (pH 7.4) containing 0.05% Tween-20; EA, antibody-sensitized sheep erythrocytes; E, sheep erythrocytes; E-M, mannan-coated sheep erythrocytes; E-M-MBL, mannan-coated sheep erythrocytes sensitized with mannan-binding lectin; EAC4, C4-coated indicator cells; EAC4-M, C4- and mannan-coated indicator cells; Tmax, time of maximal formation of enzymatically active C42 sites on EA; CH50, titer of serum lysing 50% of EA in GVB2+ via the classical pathway; AH50, titer of serum lysing 50% of rabbit erythrocytes in Mg-EGTA via the alternative pathway; LH50, titer of serum lysing 50% of mannan-binding lectin-sensitized mannan-coated sheep erythrocytes in Mg-EGTA via the lectin pathway under standardized conditions.
Received for publication October 7, 1997. Accepted for publication November 24, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and 1/50 diluted (
) depleted serum,
respectively, is shown in B.
