Division of Immunology and Cell Biology, The John Curtin School of Medical Research, and
1 Division of Biochemistry and Molecular Biology, School of Life Sciences, Faculty of Science, Australian National University, Canberra, ACT 0200, Australia
Correspondence to: C. R. Parish
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Abstract |
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Keywords: FcRI, histidine-rich glycoprotein, IgG, immune complex, monocytes/macrophages
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Introduction |
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A role for components of complement in the uptake and clearance of IC by phagocytic cells was first suggested by the demonstration that receptors for C3 exist on monocytes (11), and that large IC in blood can activate complement and both generate and incorporate C3 fragments such as C3b and C3d (12). Subsequent studies showed that blood borne C3b-opsonized IC are usually recognized by C3b receptors (CR1) on erythrocytes (3,10,13). Erythrocytes, upon entering the liver and/or spleen (14,15) may release bound IC (16) which are then free to interact with resident tissue mononuclear cells such as macrophages and monocytes via receptors for Fc, C3b and C3d (9,17,18).
Histidine-rich glycoprotein (HRG) is a plasma protein of ~80 kDa that has been shown to bind heparin (19), plasminogen (20), fibrinogen (21) and divalent metal ions (22,23), and to regulate aspects of the immune system (2428). In recent studies we also showed that HRG binds to IgG-containing soluble IC and inhibits the formation of insoluble IC in vitro by promoting the maintenance of IC in a soluble form (29). HRG also is reported to bind to macrophages, and to regulate FcR expression and phagocytosis (27,30). Since HRG incorporates in IC and binds to macrophages, it was important to examine whether HRG plays any role in the clearance of IC by mononuclear phagocytes. The present work uses flow cytometry to study the effect of HRG on the binding of IgG and IC to the human mononuclear phagocytic cell line THP1. The results show that HRG inhibits the binding of monomeric human or rabbit IgG to FcRI on THP1 cells, but promotes the binding of IC containing either human or rabbit IgG to these cells in a Zn2+-dependent manner. Collectively, the results indicate that HRG regulates the binding of both monomeric IgG and IC to mononuclear cells, and provide evidence for the existence of a complement-independent mechanism for enhancing the clearance of IC from the circulation.
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Methods |
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Purification of proteins
Native human HRG of 80 kDa mol. wt. was purified from fresh human plasma as previously described (24) by equilibrating a phosphocellulose column with loading buffer comprising 10 mM sodium phosphate (pH 6.8) containing 1 mM EDTA and 0.5 M NaCl (loading buffer). The plasma was mixed with EDTA and NaCl at the final concentrations as in the loading buffer and with [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] (ICN, Costa Mesa, CA) at 100 µg/ml and aprotinin (a trypsin inhibitor) at 2 µg/ml. The plasma was passed through the equilibrated column and unbound protein was removed by extensive washing of the column with the loading buffer. Bound HRG was then eluted from the column using the same buffer containing 2 M NaCl.
Rabbit anti-OVA IgG was purified from immunized rabbit serum by Na2SO4 precipitation and ion-exchange chromatography using a diethylaminoethyl Sephacel column. The IgG was collected from the break through peak as described (31). Using SDSPAGE analysis the purity of the HRG and IgG preparations as judged by the intensity of the contaminating bands was always >95% (29). In some experiments the purified IgG was depleted of any IgG aggregates by passage through a FPLC Superose-12 column (Pharmacia, Uppsala, Sweden), and the monomeric IgG fraction (~150 kDa) collected and used immediately.
Formation of insoluble IC
The formation of insoluble OVA:anti-OVA IgG IC was carried out by incubating rabbit polyclonal anti-OVA IgG in PBS containing 3 mM NaN3 and different amounts of HRG for 5 min in a quartz reaction vessel (final volume 1 ml) maintained at 37°C. The formation of insoluble IC was initiated by the addition of OVA at different antigen:antibody ratios. Since the absorbance due to light scattering of a suspension of particles is related to the average size of the particles, the formation of insoluble IC was monitored by measuring the absorbance of samples at 350 nm using a Varian Cary-1 spectrophotometer as described (29).
Precipitin curve analysis of IC containing rabbit IgG and OVA
Anti-OVA IgG (1 mg/ml) was mixed with different concentrations of OVA at antigen:antibody ratios in the range of 0.0050.06 (w/w) in PBS containing 10 mg/ml BSA (PBS/BSA) and 3 mM NaN3 (PBS/BSA/Az) to a final volume of 1 ml and incubated overnight at 37°C. The insoluble IC in each suspension were collected by centrifugation (104 g, 1 min), washed 3 times in PBS and then dissolved in 0.2 M NaOH. The extent of IC formation was then calculated by measuring the absorbances of each solution at 280 nm, assuming E 0.1% (w/v) = 1.4 for IgG.
FITC conjugation of IgG
Purified rabbit anti-OVA IgG (2 mg/ml) was equilibrated with coupling buffer by extensive dialysis against 0.05 M boric acid, 0.2 M NaCl (pH 9.2), and then 50 µl/ml of FITC (5 mg/ml) (Molecular Probes, Eugene, OR) dissolved in DMSO was added and the mixture incubated for 2 h at room temperature in the dark. Unreacted FITC was removed by subjecting the sample to five cycles of concentration and dilution in an Amicon ultrafiltration apparatus (Amicon, Beverly, MA). The FITCIgG was sterile filtered using a DynaGard 0.2 µm filter (Microgon, Laguna Hills, CA), before storing at 4°C until use. Based on fluorometer measurements it was estimated that approximately four molecules of FITC were coupled to each IgG molecule.
Biotinylation of proteins
HRG and human IgG were dissolved in PBS (pH 7.2) at a concentration of 1 mg/ml and then reacted with NHS-LC-biotin (1 mg/ml) (Pierce, Rockford, IL) for 30 min at room temperature. The reaction was stopped by the addition of TrisHCl buffer (pH 8.0) to a final concentration of 100 mM. Unreacted biotin was removed by washing the sample extensively (five cycles of concentration and dilution) in a Centricon 10 microconcentrator, before storing the biotinylated proteins at 20°C in small aliquots until use. Based on a competitive ELISA assay it was estimated that HRG and IgG were conjugated with approximately six and four molecules of biotin/molecule respectively.
Cell culture
The human THP1 and U937 monocyte cell lines, human Jurkat cells (CD4+ leukemic T cell line), and human MT4 cells (CD4+ T cell line) were all cultured in RPMI 1640 (Gibco, Gaithersburg, MD) supplemented with 10% heat-inactivated FCS (Commonwealth Serum Laboratories, Melbourne, Australia) and antibiotics (penicillin 120 µg/l, streptomycin 200 µg/l and neomycin 200 µg/l) at 37°C in a 5% CO2 atmosphere. The cell lines were routinely subcultured at ~0.25x106 cells/ml and allowed to grow to the maximum concentration of ~1x106 cells/ml.
Immunofluorescent flow cytometry
The cultured cells were transferred to plastic 50 ml Falcon tubes (Becton Dickinson, Lincoln Park, NJ) and centrifuged at 450 g for 5 min. Supernatants were discarded and the cells were washed 3 times in PBS. The concentration of cells was determined using a hemocytometer, and the cell suspensions pelleted and resuspended in PBS/BSA or PBS/BSA containing 20 µM Zn2+ (PBS/BSA/Zn) buffer to a final concentration of 1x107 cells/ml. The cell suspensions were then pipetted (25 µl/well) into each well of a 96-well V-bottomed plastic plate (Nunc, Roskilde, Denmark), human HRG was added at different concentrations (25 µl/well) and the mixture incubated for 1 h on ice. The cells were washed 3 times by centrifugation (800 g, 1 min) with the appropriate buffer to remove the unbound HRG, and biotinylated or FITCIgG conjugate was added in the appropriate buffer and incubated with the cells for 1 h on ice. The cells were washed 3 times by centrifugation and bound biotinylated IgG was detected by incubating the cells with FITCSTP (~ 50 µg/ml). After removing unbound FITCSTP by centrifugation, the cells were resuspended in PBS/BSA or PBS/BSA/Zn buffer (50 µl/well) and fixed by the addition of 4% paraformaldehyde in PBS (50 µl/well).
The fluorescence intensity of the cells was measured by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA). In some experiments cell populations were gated, according to the forward and side scatter, to eliminate the fluorescence deriving from dead cells, aggregated cells or large insoluble IC. The results obtained for each sample were analysed by the CellQuest program. Using this program, the median fluorescence intensity units (FIU) of each fluorescence intensity histogram were determined and plotted against the concentration of ligand or inhibitor used.
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Results |
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Effect of pre-treating THP1 cells with HRG on monomeric IgG binding
The observation that HRG binds to FcRI on THP1 cells raised the question of whether cell-bound HRG affects the ability of the cells to bind monomeric IgG via Fc
RI. To explore this possibility, THP1 cells were either pre-incubated with PBS/BSA or pre-incubated with PBS/BSA containing 2, 4 and 8 µM HRG, before washing and incubating the cells with different concentrations (40 pM to 40 nM) of monomeric bIgG. After washing away unbound bIgG the cells were then stained with FITCSTP and subjected to FACS analysis. To ensure that the bIgG was monomeric the bIgG was fractionated on a FPLC Superose-12 gel filtration column, and the monomeric IgG fraction collected and used immediately. Very similar results were obtained with both the unfractionated and the FPLC fractionated IgG preparations.
The data in Fig. 2(A) show the median FIU of cells following incubation with different IgG concentrations, and indicate that pre-incubation of THP1 cells with 2, 4 and 8 µM HRG strongly inhibited subsequent binding of IgG. In fact, at the higher concentrations of HRG (4 and 8 µM) an almost total inhibition of IgG binding was observed. Similar experiments also were carried out in which the effect of pre-incubating THP1 cells with different concentrations of HRG (9 nM to 9 µM) on the subsequent binding of a constant concentration of IgG (20 nM) was examined (Fig. 2B
). The results indicate that the pre-treatment of THP1 cells with HRG inhibits the subsequent binding of IgG and that this inhibition is dependent on the HRG concentration with HRG concentrations
2 µM inhibiting monomeric IgG binding by 90% or more (Fig. 2B
, solid squares).
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Effect of pre-incubating IgG with HRG on monomeric IgG binding to THP1 cells
The ability of HRG to bind IgG (29) suggested that the pre-incubation of IgG with HRG also might influence the binding of IgG to FcRI on THP1 cells. To explore this possibility bIgG was first pre-incubated with different concentrations of HRG in PBS/BSA and the mixture was then incubated with THP1 cells. Binding of the IgG to the cells again was assessed by FACS analysis after staining the cells with FITCSTP. As shown by the plot of IgG binding against HRG concentration (Fig. 2B
), the pre-incubation of bIgG (20 nM) with relatively low concentrations (~20280 nM) of HRG enhanced binding of the IgG to THP1 cells, whereas pre-incubation of the IgG with higher concentrations (1.129 µM) of HRG partially inhibited binding of the IgG to these cells (Fig. 2B
filled triangles). Parallel experiments also were carried out in which 20 nM bIgG was pre-incubated with different concentrations of HRG in PBS/BSA/Zn before incubating the mixture with THP1 cells. Overall Zn2+ had little or no effect on the ability of HRG to modulate the binding of IgG to THP1 cells (see Fig. 2B
, open triangles). Control experiments indicated that Zn2+ alone had no effect on the binding of IgG to THP1 cells (not shown).
The effect of HRG on the binding of IC to THP1 cells
To facilitate studies on the binding of IC to THP1 cells, FITC-labeled monomeric rabbit anti-OVA IgG (FITCIgG) was prepared and this antibody used to prepare fluorescent IC which could be used for cell binding studies. Preliminary studies showed that monomeric FITCrabbit IgG bound to THP1 cells in an almost identical manner to monomeric human IgG, i.e. near maximal binding at ~10 nM FITCIgG (not shown). Also, an analysis of the precipitin curve for IC containing OVA and FITCanti-OVA IgG indicated that maximum precipitation (~ 38% of the total added FITCIgG) occurs when the ratio of OVA:FITCIgG is between 0.02 and 0.04 (Fig. 3A, open squares). This ratio is similar to that obtained from an analysis of OVA and non-FITC-labeled anti-OVA IgG containing IC (not shown). These results suggest that the labeling of the anti-OVA IgG with FITC does not significantly affect the ability of the monomeric IgG to bind to THP1 cells or to form IC with OVA.
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Subsequent experiments investigated the effect of HRG on the binding of fluorescent OVA anti-OVA IC to THP1 cells. Pre-incubating THP1 cells with 2 µM HRG had no significant effect on the binding of IC formed with 0.5 µM FITCanti-OVA IgG at either equivalence or in antigen excess (not shown). HRG also has no effect when 20 µM Zn2+ was included in the assay. However, as with monomeric human IgG (Fig. 2), pre-treatment of the THP1 cells with HRG inhibited the binding of monomeric FITCanti-OVA IgG, an effect which was observed even when IC were present (not shown).
Our previous results (Fig. 2B, triangles) indicate that the binding of human IgG to THP1 cells is enhanced when monomeric human IgG is pre-incubated with HRG (HRG:IgG molar ratio ~110:1) before incubating the HRGIgG mixture with the cells. To determine whether HRG also enhances the binding of IC to THP1 cells, HRG was incorporated in IC by pre-incubating anti-OVA FITCIgG with HRG prior to the addition of OVA and then incubating the resultant IC with THP1 cells. In the absence of Zn2+ HRG has little or no effect on the binding of IC to THP1 cells at a wide range of HRG concentrations and at different antigen:antibody ratios (not shown). However, inclusion of 20 µM Zn2+ in the assay did result in HRG substantially enhancing the binding of IC to THP1 cells (Fig. 4
). This potentiating effect of HRG, in the presence of Zn2+, on the binding of IC to THP1 cells was seen when the HRG:IgG molar ratio approached 0.51 (Fig. 4A
). However, at higher HRG:IgG molar ratios little potentiation occurred and, in fact, HRG at a 4-fold molar excess to IgG began to inhibit IC binding (Fig. 4A
). A similar effect of the HRG:IgG molar ratio on IC binding to THP1 cells was observed when IC were formed over a wide range of antigen:antibody ratios (Fig. 4B
). Enhancement of IC binding was most marked when 0.25 and 0.5 µM HRG was incorporated in the IC (Fig. 4B
), with lower HRG concentration (
0.125 µM) being much less effective at enhancing IC binding (not shown).
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Discussion |
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A major finding of the present study is that the pre-treatment of THP1 cells with HRG significantly inhibits the binding of monomeric human IgG to THP1 cells, with the binding being inhibited ~90% by 2 µM HRG. It should be noted that this observation is physiologically relevant as the plasma concentration of human HRG is ~2 µM. Interestingly, the pre-incubation of THP1 cells with HRG also inhibited the binding of both monomeric human IgG1 and IgG3 to these cells (not shown), but the presence of Zn2+ did not alter the effect of HRG on the interaction of IgG with THP1 cells (Fig. 2B, squares). Since monomeric IgG binds to Fc
RI on THP1 cells, these findings suggest that the pre-treatment of the THP1 cells with HRG can mask the IgG-binding site on the Fc
RI. Interestingly, the presence of IC in the reaction vessel did not change the ability of HRG to inhibit the subsequent binding of monomeric IgG to THP1 cells. However, the fact that pre-treatment of the cells with HRG did not affect the binding of IC to these cells may be explained by the ability of the IC to bind to low-affinity FcR (Fc
RII, etc.) on THP1 cells which may not be blocked by HRG. Another possibility is the established ability of IC to bind to cells multivalently and hence with higher avidity, a process which may displace HRG from Fc
RI on the cell surface.
Despite the fact that pre-incubation of THP1 cells with HRG inhibited subsequent binding of monomeric IgG, the binding of IgG to THP1 cells was potentiated when IgG was pre-incubated with HRG (Fig. 2B, triangles). Similarly, the pre-incubation of HRG with monomeric human IgG1 and IgG3 also resulted in an enhanced binding of these IgG subclasses to THP1 cells (not shown). The potentiating effect of HRG under these conditions is likely to be due to the binding of the IgGHRG complexes to heparan sulfates on the cell surface via heparan sulfate binding sites on HRG (28,39). The data presented also show that the molar ratio of HRG:IgG is an important factor in determining whether HRG potentiates (HRG:IgG molar ratio ~ 110:1) or inhibits (HRG:IgG molar ratio
50:1) the binding of free IgG to Fc
RI. Thus, it would be expected that under physiological conditions plasma HRG (2 µM) would not enhance the binding of monomeric plasma IgG (~60 µM) to monocytes since the molar ratio of HRG:IgG is ~1:30.
Another major finding from the present work is that HRG potentiates the binding of IgG-containing IC to THP1 cells in the presence of physiological concentrations of Zn2+. IC containing FITCIgG and OVA were used to study the effect of HRG on the interaction of IC with THP1 cells. Turbidity assays and precipitin curve analysis of insoluble IC formed using FITCIgG and OVA indicated that insoluble IC formation in this system was indistinguishable from that which occurred when using unlabeled IgG and OVA (not shown). Also, the binding of IC to THP1 cells showed a similar pattern to the precipitin curve analysis of FITCIgG-OVA IC (see Fig 3A, cf. circles with squares). These findings indicate that the binding of IC to Fc
R is dependent on the antigen:antibody ratio of the IC mixture and that maximum binding of IC occurs at or near the equivalence antigen:antibody ratio. This result is consistent with the observation that IC formed at an equivalence antigen:antibody ratio are more effective in stimulating mouse peritoneal macrophages (40).
IC containing FITCIgG, OVA and HRG were found to bind to THP1 cells at higher levels than IC containing only FITCIgG and OVA when 20 µM Zn2+ was present. The results show, therefore, that HRG promotes the binding of IC to THP1 cells in a Zn2+-dependent fashion. There are two possible explanation for this phenomenon. First, our previous studies showed that Zn2+ substantially enhances the binding of HRG to IgG (29). Second, Zn2+ enhances the binding of HRG to heparan sulfates structures on cell surfaces (28). HRG appears to maximally potentiate the binding of IC to monocytes when the IC are formed with a HRG:IgG molar ratio of ~1. However, at higher HRG:IgG molar ratios little or no enhancement of the binding of IC occurs and, in fact, when the HRG:IgG molar ratio was 4 inhibition of IC binding was observed, presumably due to excess HRG masking FcR on monocytes.
To date the uptake of IC by the RES has been proposed to involve two mechanisms, i.e. the binding of IC to FcR and the binding of IC complexed with C3b to CR. The production of C3b or C3d requires activation of the complement cascade which results in the release of other factors (e.g. anaphylatoxins, membrane attack complex) not yet shown to be involved in the clearance of IC (3,10). The present study provides evidence for the existence of a third mechanism for enhancing the uptake of IC by monocytes, i.e. by incorporation of HRG in IC. Our results indicate that HRG can promote the binding of IC to FcR probably via binding to the glycosaminoglycan, heparan sulfate and possibly to other HRG receptors on the cell surface. The binding or incorporation of HRG into IC also may increase the avidity of these IC for the cell surface due to the formation of multivalent interactions between IC and heparan sulfate receptors for HRG.
The finding that HRG inhibits the binding of monomeric IgG to monocytes by masking FcRI is an intriguing observation. It seems likely that the region of HRG which binds IgG also binds Fc
RI since the extracellular portion of Fc
RI contains three Ig-like domains (37). Whether HRG can interact with FcR other than Fc
RI and regulate their function is currently under investigation. However, the normal physiological concentrations of HRG and IgG in plasma are such that most HRG would be associated with soluble IgG and unavailable to interact with Fc
RI.
It has been shown that the deposition of circulating IC in various organs (e.g. kidney, joint and the blood vessel wall) may lead to the development of pathological conditions such as glomerulonephritis, arthritis and vasculitis (41,42). It also has been shown that tissue IC deposits are in equilibrium with circulating IC (2,41) and that a reduction of IC in the circulation is necessary for therapy in the majority of IC-mediated diseases (43). Attenuation of the activation of complement also was proposed to be a clinical strategy to treat these pathological conditions (4447). Since HRG is a plasma protein, it is possible that in the treatment of IC-mediated diseases HRG may be used as a therapeutic agent for the prevention of the pathogenic effects of IC in terms of keeping IC in a soluble form and potentiating their uptake by the RES.
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Acknowledgments |
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Abbreviations |
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bHRG | biotinylated HRG |
bIgG | biotinylated IgG |
CR1 | complement receptor 1 |
FIU | fluorescence intensity unit |
HRG | histidine-rich glycoprotein |
IC | immune complexes |
OVA | ovalbumin |
RES | reticuloendothelial system |
STP | streptavidin |
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Notes |
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Received 12 January 1999, accepted 27 April 1999.
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References |
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