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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3898-3903
Inhibition of Activation of the Classical Pathway of Complement by
Human Neutrophil Defensins
By
Rocco H. van den Berg,
Maria C. Faber-Krol,
Sandra van Wetering,
Pieter S. Hiemstra, and
Mohamed R. Daha
From the Departments of Nephrology and Pulmonology, Leiden University
Medical Center, Leiden, The Netherlands.
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ABSTRACT |
Defensins are small, cationic antimicrobial peptides that are
present in the azurophilic granules of neutrophils. Earlier studies
have shown that defensins may influence complement activation by
specific interaction with activated C1, C1q, and C1-inhibitor. In the
present study, we show that the defensin human neutrophil peptide-1
(HNP-1) is able to inhibit activation of the classical complement
pathway by inhibition of C1q hemolytic activity. The binding site for
HNP-1 on C1q is most likely located on the collagen-like stalks, as a
clear, dose-dependent binding of HNP-1 to either intact C1q or to the
collagen-like stalks of C1q was demonstrated using enzyme-linked
immunosorbent assay (ELISA). Besides binding of HNP-1 to C1q, also a
limited binding to C1 and to a mixture of C1r and C1s was observed,
whereas no binding to C1-inhibitor was found. Because binding of HNP-1
to C1-inhibitor has been suggested in earlier studies, we also assessed
the binding of HNP-1 to mixtures of C1-inhibitor with either C1r/ C1s
or C1. No binding was found. Using a competition ELISA, it was found
that HNP-1, but not protamine, inhibited binding of biotin-labeled
HNP-1 to C1q in a dose-dependent fashion. In the fluid phase,
preincubation of HNP-1 with C1q resulted in complex formation of HNP-1
and C1q and generation of stable complexes. In conclusion, HNP-1 is
able to bind to C1q in the fluid phase and inhibits the classical
complement pathway. This mechanism may be involved in the control of an
inflammatory response in vivo.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
NEUTROPHIL DEFENSINS ARE cationic, small
cysteine and arginine-rich peptides that comprise about 40% of the
total protein content of azurophilic granules.1 Members of
the  defensin subfamily of defensins include human neutrophil
peptide (HNP) 1-3 and the less abundant HNP-4. Recently, the defensins human defensin (HS)-5 and HD-6 were demonstrated
to be present in Paneth cells.2 Two members of the human
 defensins have been described to date: human beta-defensin
(hBD)-1, expressed in various epithelia,3 and
hBD-2 that was shown to be expressed in keratinocytes and also in the
lung.4 In addition to the well established antimicrobial activity against bacteria, fungi, viruses, and parasites,5 neutrophil defensins also display other activities such as the induction of histamine release by mast cells6 and
chemotactic activity for monocytes and T cells.7,8 Also
cytotoxic activity to various autologous cells has been
described.9 Serum contains various defensin binding
molecules including serpins, 2-macroglobulin and specific complement
components that may inhibit the cytolytic activity.10-12
With respect to complement, predominantly binding to the complex of the
first component C1, C1q and C1-inhibitor was
demonstrated.10,13 Complex formation between defensins and
C1 not only may inhibit the cytolytic activity of defensins as
suggested before,10 but potentially could also inhibit the activity of C1. C1, composed of C1q, C1r, and C1s, is not only able to
initiate activation of the classical pathway of complement, but C1
subcomponents like C1q are also able to activate various cells via
interaction with specific cellular receptors. Once C1, bound to an
activator via the globular heads of C1q, is activated, C1-inhibitor is
able to dissociate C1r/C1s from C1q.14 C1q, still bound to
the activator, but now with a free collagen-like stalk, is able to
activate different cell types via receptors that are specific for C1q
(C1qR). Three types of C1qR have been described; the receptor for the
globular domain of C1q (gC1qR15,16), the receptor for the
collagen-like stalks of C1q, which has high homology with Calreticulin
(cC1qR/CaR17-19), and the receptor for the
collagen-like stalks that induces phagocytosis by neutrophils (C1qRp20-22). We have shown earlier that cC1qR/CaR inhibits
C1q hemolytic activity by binding to the collagen-like stalks of
C1q.23 In this report, we describe the capacity of HNP-1 to
specifically bind to the collagen-like stalks of C1q, resulting in
inhibition of C1q hemolytic activity. Because defensins are a major
constituent of neutrophil azurophilic granules, we suggest that upon
degranulation of neutrophils, inhibition of complement activity may
occur in the microenvironment of the neutrophil.
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MATERIALS AND METHODS |
Isolation of HNP-1.
HNP-1 was isolated from neutrophils as described by Van Wetering et
al.24 In short, neutrophils were disrupted by nitrogen cavitation and after centrifugation, granules were obtained from the
supernatant. After extracting the granules with 5% acetic acid, the
extracts were fractionated using gel filtration on Sephacryl S-200 HR
columns (Pharmacia, Roosendaal, The Netherlands) and reverse phase high-performance liquid chromatography
(HPLC) on C18 (Vydac, The Separations Group, Hesperia,
CA). The preparations were analyzed by tricine sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gold-PAGE (AU
PAGE), and laser desorption mass spectrometry (Lasermat;
Finnigan MAT, Hemel Hempstead, UK) and were shown to be devoid of other
contaminating proteins.24 An aliquot of purified HNP-1 was
biotinylated with Biotin-N-Hydroxysuccinimide ester
(HNP-1biotin) according to the manufacturer's protocol
(Zymed Laboratories Inc, San Francisco, CA).
Isolation of C1 and C1q.
C1 and C1q were isolated as described earlier.23 C1
from human serum was precipitated with PEG-6000 (E. Merck,
Amsterdam, The Netherlands) with a final concentration of 3% (wt/vol).
The pellet was dissolved in Veronal-buffered saline, pH 7.4, conductivity was adjusted to 12 milliSiemens (mS),
and EDTA to a final concentration of 2 mmol/L was added. After
centrifugation, the solution was applied to a rabbit IgG-Sepharose
column and after washing, bound C1q was eluted with a step gradient of
1 mol/L NaCl containing 2 mmol/L EDTA. Fractions with C1q hemolytic
activity23 were pooled, concentrated, and fractionated on a
Superdex 200 Hiload 26/60 FPLC gel filtration column (Pharmacia).
Again, the fractions were tested for functional C1q activity and
positive fractions were pooled, concentrated to 8 mg/mL and stored on
ice. An aliquot was analyzed by SDS-PAGE and found to be devoid of
other contaminating proteins. In enzyme-linked immunosorbent assay
(ELISA), no detectable C1-inhibitor, C1r, or C1s was
found.25
Preparation of C1q globular heads and C1q collagen-like stalks.
For the preparation of C1q globular heads, C1q was incubated for 20 hours at 37°C with collagenase type Ia (Sigma Chemical Co, St
Louis, MO) in 50 mmol/L Tris-HCl with 10 mmol/L CaCl2, pH
7.2. The material was then fractionated by gel filtration HPLC on a TSK
3000 SW column (LKB Producter AB, Bromma, Sweden) and finally analyzed
on SDS-PAGE. The 30-kD protein was found to be devoid of intact
C1q.26
Effect of HNP-1 on complement activity.
To determine the effect of HNP-1 on complement activity, standard
assays as described earlier27-30 for either CH50, AP50, or C3 were performed in the presence of increasing concentrations of
HNP-1.
Effect of HNP-1 on C1q hemolytic activity.
To determine the effect of HNP-1 on C1q hemolytic activity, a hemolytic
assay described earlier23 was used. In short,
1 × 107 sheep erythrocytes sensitized with rabbit
antibodies (EA) were incubated for 1 hour at 37°C with 1/25 diluted
C1q depleted serum (C1qD), a limiting dose of C1q, and increasing
concentrations of HNP-1. After incubation for 1 hour at 37°C, the
reaction was stopped by the addition of 1.5 mL phosphate-buffered
saline (PBS). The percentage lysis was determined relative to a reagent
blank and 100% lysis, expressed as units/mL (Z) and converted to
percentage inhibition.
ELISAs for detection of binding of HNP-1 to complement proteins.
Ninety-six well microtiter plates (Greiner BV, Alphen a/d Rijn, The
Netherlands) were coated overnight at room temperature with 1 µg amounts of C1q, C1q globular heads, C1q
collagen-like stalks, or bovine serum albumin (BSA) in coating buffer
(0.1 mol/L sodium carbonate, pH 9.6). All reaction volumes were 100 µL. Alternatively, plates were coated in the same buffer with
increasing concentrations of either C1, C1q, mixtures of C1r and C1s,
C1-inhibitor, or BSA. Also mixtures, consisting of different
concentrations of either C1r/C1s and C1-inhibitor or C1 and
C1-inhibitor were used as coating protein. After washing, various
concentrations of HNP-1Biotin were added to the wells in
PBS containing 0.05% (vol/vol) Tween and 1% delta-fetal calf serum
(FCS). After 1 hour of incubation at 37°C, the wells
were washed and bound HNP-1Biotin detected with
streptavidin-horseradish peroxidase (HRP). After washing,
ABTS (2,2 -azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)
(Sigma Chemicals Co) was added as a substrate for HRP. The optical
density at 415 nm was measured using a Microplate Biokinetics Reader EL
312e (Bio-tek Instruments Inc, Winooski, VT).
To determine binding specificity, a suboptimal concentration of
biotinylated HNP-1 was preincubated for 30 minutes at 4°C with
increasing concentrations of protamine or unlabelled HNP-1 in PBS/Tween
before it was added to C1q-coated microtiter plates and incubated for 1 hour at 37°C. After washing, bound biotinylated HNP-1 was detected
by a 1-hour incubation with streptavidin-HRP, washing, and subsequent
staining with ABTS.
Generation of fluid phase C1q/HNP-1 complexes.
To determine whether interaction of C1q and HNP-1 resulted in the
formation of complexes of these two proteins, 7 µg C1q and 50 µg
HNP-1 were incubated for 30 minutes at 4°C in PBS and subsequently applied to a Sephadex G-150 column (Pharmacia Biotech, Roosendaal, The
Netherlands) in PBS. As controls, C1q alone and HNP-1 alone were also
run on the same column. In the fractions, C1q antigen and HNP-1 antigen
were assessed by ELISA as described above.
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RESULTS |
To determine whether HNP-1 had an effect on the classical complement
pathway, EA in 0.5% (vol/vol) normal serum was incubated with various
concentrations of HNP-1 for 1 hour at 37°C and assessed for
hemolysis of EA. HNP-1 inhibited the CH50 activity of normal serum
(2.72 U/mL) in a dose-dependent manner (Fig
1A). To find out whether HNP-1 also influenced AP50 activity, rabbit
erythrocytes, together with 5% (vol/vol) normal serum in the presence
of various concentrations of HNP-1 were incubated for 1 hour at
37°C and assessed for hemolysis. HNP-1 had no detectable effect on
AP50 activity. Taken together with the effect of HNP-1 on CH50
activity, the results suggested an effect of HNP-1 early in the
classical pathway. Therefore, 0.5% (vol/vol) serum was incubated with
various concentrations of HNP-1 for 1 hour at 37°C and subsequently
analyzed for residual C1q and C3 hemolytic activity. The presence of
HNP-1 resulted in a dose-dependent inhibition of C1q hemolytic
activity, while no effect on C3 (Fig 1B) or on C2 and C4 was found
(data not shown).
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| Fig 1.
HNP inhibits C1q hemolytic activity and CH50. To
determine the effect of HNP-1 on CH50, EA diluted in normal serum, were
incubated with various concentrations of HNP-1 and assessed for
hemolysis. Alternatively, AP50 was determined by incubation of rabbit
erythrocytes in the presence of different concentrations of HNP-1 (A).
Also, the effect of HNP-1 on specific complement components was
determined (B). Dilutions of serum were incubated with increasing
concentrations of HNP-1 and after incubation, the hemolytic activity of
either C1q or C3 was determined. The data are expressed as the
percentage inhibition of lysis, compared with values obtained in the
absence of HNP-1.
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The results above indicated a direct interaction of HNP-1 with C1q.
Therefore, ELISA wells were coated with fixed concentrations of C1q,
C1q globular heads, C1q collagen-like stalks, or BSA and assessed for
binding of HNP-1Biotin. The results
(Fig 2) indicate that HNP-1 binds to intact
C1q and to C1q collagen-like stalks in a dose-dependent fashion,
whereas only minimal binding to C1q globular heads was observed.
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| Fig 2.
Dose-dependent binding of HNP-1 to intact C1q ( ), C1q
collagen-like stalks ( ), and C1q globular heads ( ). ELISA wells
were coated with 1 µg of either C1q, C1q globular heads, C1q
collagen-like stalks, or BSA ( ) and incubated with increasing
concentrations of HNP-1Biotin. After incubation, bound
HNP-1Biotin was determined by subsequent incubation with
streptavidin-HRP and ABTS.
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Earlier studies have shown a specific interaction of HNP-1 with
C1-inhibitor/C1r/C1s complexes and with C1-inhibitor/C1
complexes.10 Therefore, ELISA wells were coated with fixed
concentrations of C1q, C1r/C1s, C1-inhibitor, or intact C1 and
incubated with increasing concentrations of HNP-1Biotin.
BSA was used as control. There was a dose-dependent binding of
HNP-1Biotin to C1q (Fig 3) and,
to a limited extent, to C1r/C1s and C1. The binding to the C1r/C1s
preparation may be explained by a 1% (wt/wt) contamination of the
preparation with C1q. No significant binding of HNP-1 to C1-inhibitor
was observed.
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| Fig 3.
Binding of HNP-1 to C1q (), C1r/C1s (),
C1-inhibitor ( ), C1 (), and BSA ( ). Microtiter plates were
coated with 1 µg C1q, C1r/C1s, intact C1, C1-inhibitor, or BSA and
incubated with increasing concentrations of HNPBiotin.
After washing, bound HNPBiotin was detected by subsequent
incubation with streptavidin-HRP and ABTS. Optical density was measured
and plotted.
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In additional studies, HNP-1 was coated on ELISA wells and assessed for
binding of C1q, C1r/C1s, C1-inhibitor, and intact C1 using
HRP-conjugated polyclonal antibodies against these components. Also, in
these experiments, significant binding of HNP-1 to C1q was seen,
whereas no binding to C1-inhibitor was observed and only limited
binding to C1r/C1s and C1 (data not shown).
To investigate whether C1-inhibitor affects the binding of HNP-1 to C1
or its subcomponents, the following experiment was performed. ELISA
wells were coated with PBS, BSA, C1q, C1 or C1r/C1s alone, or with
mixtures containing C1-inhibitor. These mixtures were first
preincubated in PBS for 30 minutes at 37°C to allow interaction of
C1-inhibitor with the various components. After coating to ELISA wells,
HNP-1Biotin was added and bound HNP-1Biotin
detected with streptavidin-HRP (Fig 4). As
before, clear binding of HNP-1 to C1q was observed with no change in
binding in the presence of C1-inhibitor. Binding of
HNP-1Biotin to C1-inhibitor was detectable, however,
clearly less than to C1q.
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| Fig 4.
Effect of complex formation with C1-inhibitor for HNP-1
binding. ELISA wells were coated with 1 µg BSA, C1-inhibitor, C1q,
intact C1, or C1r/C1s. In addition, the same concentration of these
proteins was coated after preincubation with 1 µg of C1-inhibitor.
Subsequently, after washing, a fixed amount of HNP-1Biotin
was added and assessed for binding. After washing, bound
HNP-1Biotin was determined by incubations with
streptavidin-HRP and ABTS.
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It is possible that the interaction of HNP-1 with C1q is dependent on
charge interactions. Therefore, ELISA wells were coated with a fixed
concentration of C1q and subsequently incubated with a fixed
concentration of HNP-1Biotin in the presence of various
concentrations of protamine or unlabelled HNP-1. While unlabelled HNP-1
dose-dependently inhibited the binding of HNP-1Biotin to
C1q, no detectable effect of protamine was found
(Fig 5).
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| Fig 5.
Specific binding of HNP-1 to C1q. A fixed concentration
of HNP-1Biotin was incubated with increasing concentrations
of either unlabelled HNP-1 () or Protamine () for 30 minutes at
4°C, and then added to microtiter wells that were coated with 1 µg C1q. After incubation, bound HNP-1Biotin was detected
as described. The optical density was measured and compared with
binding of HNPBiotin that was not preincubated. The
percentage inhibition of binding induced by HNP or protamine is
depicted.
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Although HNP-1 was shown to bind to solid phase bound C1q and vice
versa, it is possible that binding to neoepitopes, which appear after
coating of the protein, may be involved in the observed interaction. To
demonstrate binding of C1q to HNP-1 in the fluid phase, the following
experiment was performed. C1q and HNP-1 alone or HNP-1 preincubated
with C1q were fractionated on a gel filtration column. In the
fractions, HNP-1 and C1q were determined by specific ELISAs. The
profiles of the gel filtration columns are shown in Fig 6. After filtration of HNP-1 alone,
HNP-1 is mainly retrieved in the later fractions (fraction 8), whereas
filtration of HNP-1, preincubated with C1q, was mainly retrieved in the
early fractions. In the same early fractions, also C1q could be
retrieved when filtered in the absence of HNP-1 and when preincubated
with HNP-1. Colocalization of HNP-1 with C1q in the early fractions
clearly indicates complex formation of HNP-1 with C1q in the fluid
phase.
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| Fig 6.
Binding of HNP-1 to C1q in the fluid phase. HNP-1 (A) or
C1q (B) alone or a preincubated mixture of C1q and HNP-1 (C) was
fractionated on Sepharose G150 columns. HNP-1 () and C1q () were
detected by ELISA.
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DISCUSSION |
This report describes the binding of HNP-1 to the first subcomponent of
complement C1q. Binding to C1q was dose-dependent, not solely dependent
on charge interactions, and specific, as was demonstrated by
competition studies. Two earlier studies described binding of HNP-1 to
components of the C1-complex.10,13 Panyutich et
al,10 using Western blotting of purified complement
components, described that HNP-1 binds either to complexes of activated
C1 and C1-inhibitor or to complexes of C1-inhibitor and activated C1s.
No binding was observed to individual preparations of C1q, C1-inhibitor, or C1. In the present study, we determined binding of
HNP-1 to purified C1, C1q, C1r/C1s, C1-inhibitor, and complexes of
C1-inhibitor with C1 using ELISA. In agreement with the study of
Panyutich et al, only limited binding of HNP-1 to either purified C1 or
purified C1-inhibitor was detected. Also a limited binding of HNP-1 to
C1r/C1s was found. In contrast with the study of Panyutich et al,
however, marked binding of HNP-1 to highly purified C1q was observed.
HNP-1 was shown to bind to immobilized C1q and vice versa. Also
HNP-1/C1q complex formation in the fluid phase was clearly detected
using gel filtration. These experiments show that the C1q subcomponent
of C1, without denaturation or association to C1r/C1s or C1-inhibitor,
is able to bind avidly to HNP-1. The discrepancy between our findings
and the report of Panyutich et al may be explained by the
interpretation of their results. C1q was not able to enter the gel upon
native PAGE and therefore an interaction of HNP-1 with C1q could not be
visualized. However, labelled protein was clearly present in the slots
of the gels, which might reflect the presence of HNP-1/C1q
precipitates.
C1q epitopes are fully exposed after C1q is dissociated from C1r and
C1s present in the complex of activated C1. Dissociation of C1q occurs
in vivo after inactivation of C1 by C1-inhibitor. The finding that
inactivation of activated C1 by C1-inhibitor is necessary for binding
to HNP-1, is in agreement with our findings on the binding of C1q to
HNP-1.
More recently Prohaszka et al13 demonstrated that
defensins, fixed on ELISA plates, are able to bind C1q in a
dose-dependent fashion, confirming our findings. However in contrast to
our report, they also describe defensin-induced activation of the
classical pathway of complement, as assessed by deposition of C4b. The
difference between our studies and those of Prohaszka et al is that in
our studies we used defensins in the fluid phase while Prohaszka et al
used defensins that had been immobilized on solid phase ELISA wells. It
is possible that binding of defensins to a solid phase results in the
exposure of C1q binding sites that are not available in fluid phase
defensins and that are able to capture C1q in a different fashion than
fluid phase defensin. Thus, captured C1q may still be able to activate
the classical pathway of complement. Alternatively, immobilized
defensins may have bound aggregates of C1q leaving C1q molecules in the
aggregate that had not interacted with defensins available for
complement activations. A number of findings in the study of Prohaszka
et al, however, cannot be fully explained by the hypothesis that
defensin induces classical pathway activation. For example 5 µg/mL of
solid phase defensin was shown to induce marked C4b deposition in the
presence of Mg/EGTA and even in heated human serum, in which also the
alternative pathway is not operative. Therefore, at least part of the
C4b deposition on defensin-coated microwells, as observed by Prohaszka et al, may not result from complement activation, but may result from
other, possibly nonspecific, binding phenomena.
It has been described that receptors for the collagen-like domains of
C1q can specifically inhibit the classical complement pathway by
preventing the binding of C1r and C1s to C1q, which is required for
formation of an intact C1 complex.23 Earlier we described a
bacterial C1q binding protein (C1qBP) that is able to dissociate
preformed C1.31 Inhibition of C1q hemolytic activity can
also occur by binding of C1q to specific receptors for the globular
heads of C1q (gC1qR). Inhibition of hemolytic activity in this case is
based on the fact that C1q is no longer able to bind a complement
activator with its globular head.15 The present report
describes that the binding of HNP-1 to C1q also prevents activation of
the classical pathway by interference at the level of C1q. Because the
collagen-like stalks of C1q are the binding site for HNP-1 on C1q, this
might explain the very limited extent of binding of HNP-1 to intact C1,
in which the stalks of C1q are only partially exposed.32 In
sepsis, characterized by massive neutrophil degranulation and high
circulating defensin levels,33 this process might be
responsible for the observed inactivation of the complement
system.34-37
Complex formation of HNP-1 with C1q might also increase the turnover
rate of both proteins. The activity of cytotoxic defensins in serum is
known to be controlled by binding of HNP-1 to serum proteins including
the F-form of -2-macroglobulin11 and members of the
serpin family of serine protease inhibitors.12 Despite the
high concentrations of these proteins in full blood, HNP-1 at
concentrations well below those observations in septic plasma, was able
to inhibit C1q complement activity. Presumably this indicates that the
association rate of HNP-1 with C1q is higher than that of HNP-1 with
other plasma proteins. However, other mechanisms are also possible.
Because of the presence of cC1qR/CaR on both lymphoid and nonlymphoid
cells, it is possible that binding of HNP-1 to C1q facilitates
clearance of cytotoxic HNP-1 via this receptor. Preliminary experiments
by Panyutich et al10 in which uptake of
HNP-1/C1s/C1-inhibitor complexes by the human hepatoma HepG2 cell line
was shown, support this hypothesis. Another possibility however is that
defensins might be cleared from the fluid phase by binding to the cell
surface of, eg, macrophages or neutrophils, just because of their
cationic nature. This has been described earlier for other cationic
proteins derived from the azurophilic granules of neutrophils, such as
myeloperoxidase and proteinase 3.38,39 Because C1qR of
different types are expressed on the surface of various cell types, we
hypothesize that HNP-1, in complex with C1q, is cleared from the
circulation by binding to cell surface expressed C1qR.
In addition to inhibiting activation of the classical complement
pathway as observed in the present study, defensins have also been
reported to inhibit fibrinolysis.40 This effect was suggested to be the result of defensin-mediated shielding of
plasminogen that is bound to fibrin from activation by tissue type
plasminogen activation.41 Whether binding to C1q inhibits
this or other effects of defensins, such as inactivation of
serpins,12 remains to be determined. In conclusion, complex
formation of HNP-1 and C1q can result either in inhibition of an
inflammatory reaction by inhibiting the hemolytic activity of C1q or
could result in clearance of HNP-1 via specific C1qR on the cell
surfaces of many cell types. Together all of these mechanisms are
involved in the downregulation of both complement and neutrophil
activation. Therefore, C1q deficiency, in addition to -1 proteinase
inhibitor deficiency,12 might result in impaired regulation
of defensin-mediated effects.
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FOOTNOTES |
Submitted February 19, 1998;
accepted July 6, 1998.
Supported by the Netherlands Organization for scientific research and
by a grant from the Dutch Asthma Foundation (93.61).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mohamed R. Daha, PhD,
Department of Nephrology, Building 1, C3-P, Leiden University Hospital,
PO Box 9600, 2300 RC Leiden, The Netherlands;
e-mail:M.R.Daha{at}Nephrology.Medfac.Leidenuniv.nl.
| |
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Abstract
Introduction