| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of Rheumatology and Clinical Immunology, Teresa Scheu Rheumatoid Arthritis Research Laboratory, Department of Internal Medicine, University of Arkansas for Medical Sciences, and Veterans Administration Medical Center, Little Rock, AR 72205
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RI; activation of polymorphonuclear neutrophils; effect
on the Ab-combining site of Abs; and in vivo inflammatory activity in a
rabbit model of acute arthritis. Rheumatoid synovial fluids contained
significantly greater concentrations of nitrosated and chlorinated IgG
compared with ostearthritic specimens. In vitro exposure of human IgG
to HOCl and ONOO resulted in a concentration-dependent decrease in C3
and C1q fixation. The decrease in Fc domain-dependent biologic
functions was confirmed by competitive binding studies to the FcRI
of U937 cells. HOCl-treated IgG monomer was 10 times less effective in
competing for binding compared with native IgG, and ONOO-treated IgG
was 2.5 times less effective. The modified IgGs were also ineffective
in inducing synthesis of H2O2 by human PMN. The
Ag-binding domains of IgG also showed a concentration-dependent
decrease in binding to Ag. The ability of the modified IgGs to induce
acute inflammation in rabbit knees decreased 20-fold as gauged by the
intensity of the inflammatory cell exudates. These studies clarify the
modulating role of biological oxidants in inflammatory processes in
which Ag-autoantibody reactions and immune complex pathogenesis may
play an important role. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As is the case with oxygen free radicals, NO and its derivatives exhibit both proinflammatory and antiinflammatory activities (1, 7). We and others have shown that the superficial chondrocytes secrete large amounts of NO (8, 9), which can react with oxygen radicals derived from inflammatory cells and chondrocytes at the cartilage surface, to form peroxynitrite (ONOO) and other reactive molecules able to nitrosate and aggregate IgG (10). Although the modified IgG may be aggregated, its capacity to induce inflammation compared with similar "native" immunocomplexes is not known.
In the present studies, we have confirmed the presence of chlorinated and nitrosated IgG species in synovial fluids. We also show that the IgG aggregates modified by the reactive radicals and NO have a markedly diminished proinflammatory capacity, both in vitro and in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human IgG was purified from normal serum by affinity chromatography with Hitrap Protein A columns (Pharmacia Biotech, Piscataway, NJ). Mouse monoclonal anti-nitrotyrosine was purchased from Upstate Biotechnologies (Waltham, MA). Affinity-purified rabbit anti-human C3 was purchased from Dako (Carpinteria, CA). Rabbit anti-human C1q antiserum was obtained by immunizing rabbits with purified human C1q (11). Rabbit anti-BSA Abs were purified as described previously (12). Affinity-purified goat anti-human IgG, anti-rabbit IgG, and anti-mouse IgG Abs were purchased from BioSource International (Camarillo, CA) and labeled with [125I]Na with IODO-beads (Pierce, Rockford, IL). Rabbit anti-HOCl-treated IgG antiserum was prepared by immunization of rabbits with human IgG treated four times with 0.6 mM HOCl in CFA. The Abs were purified sequentially by affinity chromatography using Sepharose 4B-HOCl-IgG and Sepharose 4B-IgG columns. The resulting Abs were shown to react only with HOCl-IgG by RIA. No reactivity was detected against normal IgG or HOCl-treated BSA (results not shown). HOCl was distilled from 5% sodium hypochlorite (Sigma, St. Louis, MO) as previously described (5). ONOO was synthesized from NaNO2 and hydrogen peroxide (H2O2) by the method of Rakesh (13). Purified human myeloperoxidase was a gift from Dr. Issac Ginsburg (Hebrew University, Hadassah School of Dental Medicine, Jerusalem, Israel) (5). Synovial fluids from patients with rheumatoid arthritis (RA), osteoarthritis, or gout were obtained as a byproduct of diagnostic or therapeutic knee aspirations. The fluids were centrifuged at 2000 rpm for 15 min to remove cells before storage at -80°C.
Chlorination and nitrosation of IgG
HOCl solutions (20–100 mM) were prepared by diluting HOCl with distilled water to final concentrations of 0.2–1.0 mM. The HOCl solutions were added to affinity-purified human IgG, 1.0 mg/ml. The HOCl-IgG solutions were incubated at 37°C for 1 h. Nitrosation was performed in a similar fashion by adding ONOO solutions of different concentrations to human IgG.
Quantitation of nitrosated IgG in synovial fluids
Synovial fluid IgG was purified by absorption of 1 ml volumes onto protein A affinity columns (Hitrap Protein A) following brief incubation with testicular hyaluronidase (100 µg/ml). Monoclonal anti-nitrotyrosine Ab at 1:500 dilution was coated on polyvinyl chloride microtiter 96-well plates. The wells were washed and blocked with PBS-10% FCS. Triplicate wells were then incubated with the synovial fluid IgGs, washed, and incubated with 125I-labeled goat anti-human IgG. After washing, the bound radioactivity was measured in a COBRA-2 gamma scintillation spectrometer (Packard, Meriden, CT). Wells containing mouse IgG of the same isotype were used for each synovial fluid for background control. Total IgG in each sample was determined by RIA using normal human IgG to construct a standard curve.
Quantitation of chlorinated IgG in synovial fluids
Affinity-purified rabbit anti HOCl-IgG (3.0 µg/ml) was coated on microtiter wells. The wells were washed and blocked with PBS-1% normal rabbit serum. Triplicate wells were then incubated with the synovial fluids at 1:100 dilution containing 0.05 M DTT, washed with PBS-normal rabbit serum, and incubated with 125I-labeled goat anti-human IgG. Wells containing normal rabbit IgG were used for each synovial fluid as background controls. Total IgG concentration in each fluid was measured by RIA.
Polymorphonuclear leukocyte (PMN) isolation
PMN were isolated from healthy donors by density gradient centrifugation with PolymorphPrep (Accurate Chemical and Scientific, Westbury, NY). Contaminating erythrocytes were lysed with 0.2% NaCl. The PMN were washed and resuspended in a solution containing140 mM NaCl, 10 mM KH2PO4, 5.5 mM dextrose, 0.28 mM phenol red, HRP 8.5 U/ml, pH 7.0, and used immediately after isolation. PMN viability was >95% by trypan blue staining.
Colorimetric assay for H2O2
Microtiter plates were coated with normal or oxidized IgGs as
described above. Preliminary experiments using
125I-labeled anti-human IgG
Ab indicated that the amounts of treated IgG attached to the plastic
wells was identical with that found for native IgG (results not shown).
After twice washing and blocking with PBS containing 0.1% BSA, 100
µl of 1.0 x 106/ml PMN suspension in the
buffer described above containing 1.0 mM CaCl2
were added to each well. After 1 h incubation at 37°C,
H2O2 was measured
colorimetrically (oxidized phenol red) (14). PMA (10
ng/ml) was added to some wells as a positive control. Standard curves
were constructed with seven dilutions of 30%
H2O2. As a rule,
plastic-bound normal human IgG resulted in
H2O2 secretion of 
3.0
nmol/1.0 x 105 cells; about 50% of the
H2O2 secretion induced by
PMA stimulation.
C1q and C3 binding to normal and oxidized IgG
Normal and oxidized IgG-coated microtiter plates were washed and blocked with PBS containing 0.2% Tween 20. After incubation with 50 µl 1:10 dilutions of normal or heat-inactivated normal human serum for 1 h at 37°C, followed by washing, 50 µl of 2 µg/ml rabbit anti-C3 or C1q were added and incubated for 2 h at room temperature. After washing, 125I-labeled goat anti-rabbit IgG was added and incubated for 2 h. After washing, bound radioactivity was measured in a gamma spectrometer.
Fc receptor (FcRI) binding assay
Experiments to gauge the capacity of the oxidized IgG monomers to compete for binding to FcRI by normal IgG were conducted by a modified method of Crabtree et al. (15). Briefly, normal and oxidized IgG monomers were obtained by gel filtration on Sepharose CL-6B columns. 125I-labeled normal IgG was added at saturation (50 nM) to 1.5 x 106 U937 cells (CRL-1593.2; American Type Culture Collection, Manassas, VA) in medium containing 5 µg/ml cytochalasin B with increasing concentrations (0–500 nM) of unlabeled normal, HOCl, or ONOO-treated IgG monomers. After incubation at 37°C for 1 h, the free IgG was separated from the bound molecules by centrifugation of the cells through a mixture of 16% paraffin oil (Fisher Scientific, Pittsburg, PA) and 84% silicon oil (Sigma) at 9000 rpm for 1 min. An aliquot of the supernatant and the tips of the conical tubes containing the cell pellets were cut off, and the free and cell-bound radioactivities were measured in a gamma scintillation spectrometer.
Ag binding of oxidized Ab
Oxidation of 1.0 mg/ml affinity-purified rabbit anti-BSA with increasing concentrations of HOCl or ONOO was performed as described above for human IgG. BSA (20 µg/ml) was added to microtiter wells and incubated for 2 h. After washing and blocking, 50 µl of normal or oxidized rabbit anti-BSA Abs (100 ng/ml) were added to the plates and incubated for 2 h. After washing and blocking, 125I-labeled goat anti-rabbit IgG was added and incubated for 2 h. After washing, bound radioactivity was measured in a gamma spectrometer.
Acute arthritis in rabbits
New Zealand White rabbits of either sex (2.0–2.5 kg) were used to induce acute arthritis by intraarticular injection in the knee joints of 100 µg of heat aggregated, HOCl (0.8 mM), or ONOO (0.8 mM)-treated IgG in 1 ml sterile saline solution or vehicle alone. Aggregates from the three types of IgG of identical molecular masses were isolated by gel chromatography using Sepharose CL-6B columns (Pharmacia). The fractions containing high molecular mass aggregates were pooled, concentrated, and sterilized by filtration. Preliminary experiments using heat-aggregated IgG in increasing concentrations (10, 100, and 1000 µg) indicated that near maximal acute inflammatory responses were obtained with a concentration of 100 µg per joint. The animals were sacrificed 48 h after the intraarticular injection, the knee joints were opened, and the suprapatellar pouches were flushed with 1 ml PBS containing heparin (16). The cells were counted manually in a modified Neubauer chamber.
Statistical analysis
Statistical analysis was performed using the unpaired Students
t test (StatView version 4.5; SAS Institute, Cary, NC).
Values of p 
0.05 were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
HOCl-IgG was quantitated in 27 rheumatoid, 18 osteoarthritic, and
9 gouty synovial fluids. As shown in Fig. 1
A, HOCl-IgG concentrations
were highest in the rheumatoid fluids (80.5 ± 3.1 µg/ml)
compared with osteoarthritic (66.6 ± 1.8 µg/ml;
p < 0.002), and gouty fluids (63.1 ± 3.4
µg/ml; p < 0.005 with respect to RA). Of interest
was the finding that when the values were corrected for IgG
concentrations in each synovial fluid, the mean percentages of HOCl-IgG
in rheumatoid and osteoarthritic fluids were similar (0.9 ± 0.08
vs 1.0 ± 0.08%; p > 0.2), with the mean
percentage in gouty fluids being significantly lower (0.6 ±
0.1%).
|
Measurements of nitrosated IgG were carried in 29 rheumatoid and
19 osteoarthritis synovial fluids. Fig. 1
B shows the results
obtained without correction for the IgG concentration in each synovial
fluid. The rheumatoid fluids contained a mean of 12.1 ± 1.4
µg/ml, whereas the osteoarthritis specimens had 5.8 ± 0.8
µg/ml (p < 0.002). When the results were
expressed as percent of the total IgG in each fluid the difference was
still statistically significant (p < 0.04),
albeit not as considerable as that seen with the uncorrected
values.
Complement C3 and C1q binding to modified IgGs
The capacity of HOCl-treated IgG to fix the complement components
C3 and C1q was measured by incubation of fresh human serum as a source
of complement with plastic-bound IgG incubated with increasing
concentrations of HOCl or ONOO. Preliminary experiments were conducted
to show that the degree of binding of the modified IgGs to the plastic
wells was not decreased with treatment (results not shown). The results
in Fig. 2A indicate that
incubation of IgG with HOCl resulted in a significant
concentration-dependent decrease in C3 fixation, which became almost
negligible at 0.6 mM concentration. Treatment of IgG with ONOO also
resulted in a decrease in C3 binding, albeit not as pronounced as that
shown for HOCL (Fig. 2B). Similar results were obtained in
experiments where C1q binding to IgG was studied (Fig. 3, A and B). In the
case of ONOO treatment, the decrease in C1q binding was more pronounced
at low concentrations, and it did not decrease further as the ONOO
concentration was increased.
|
|
The results described above suggested that oxidant attack altered
the physicochemical characteristics of the Fc domain of IgG. Many of
the important cell-modifying functions of IgG are mediated by its
binding to membrane-bound Fc receptors present in a variety of
inflammatory and immune cells (17). Therefore, we
conducted experiments to study the binding properties of the modified
IgGs to FcRI in the myelo-monocytic cell line U937. In these studies,
we measured the ability of the modified IgG monomers to compete with
native IgG for binding to the cell receptor. As shown in Fig. 4, A and B, 1:1
mixing of labeled and unlabeled native IgG reduced
125I-IgG binding by an expected 50%, whereas it
took 10-fold more HOCl-IgG and 2.5-fold more ONOO-treated IgGs to
achieve the same degree of competition.
|
The significant reduction in FcR binding by the modified IgGs
suggested that they may also have a decreased capacity to induce cell
activation. Normal human PMN were incubated with plastic-bound normal
and modified IgGs. H2O2
production was measured as an indicator of activation. Preliminary
experiments showed that the magnitude of native and modified IgGs
binding to the plastic wells was identical. As shown in Fig. 5, A and B, there
was a dose-dependent decrease in
H2O2 production as the
oxidants’ concentrations increased, which in the case of HOCl reached
baseline levels with IgG treated with 0.8 mM.
|
The experiments described above probed the consequences of
oxidative modification of the Fc portion of the IgG molecules.
Additional experiments were conducted to study the effects of oxidative
attack on the Ag-binding domain of affinity-purified rabbit
anti-BSA IgG. Exposure of the affinity-purified Ab to HOCl resulted
in a concentration-dependent loss of binding capacity (Fig. 6A). Of particular interest
was the observation that the rabbit Ab was particularly vulnerable to
attack by ONOO. As seen in Fig. 6B, exposure to as little as
0.4 mM concentrations reduced binding capacity by almost 90%.
|
Taken as a whole, the in vitro results described thus far strongly
suggested that the modified IgGs may have significantly reduced
capacity to induce inflammation. However, it was deemed necessary to
study the proinflammatory properties of the modified IgGs in an in vivo
model of inflammation, and in particular a model of inflammatory
arthritis. Fig. 7 shows the results
obtained using the magnitude of cellular inflammatory responses of
rabbit knee joints as an index of the intensity of inflammation. Extra
care was taken to isolate aggregates of similar molecular masses for
the heat-, HOCl-, or ONOO-treated IgGs. Whereas injection of
heat-aggregated IgG induced a mean response of 4.5 ± 1.2 x
105 cells per joint, injection of the oxidized
aggregates or vehicle resulted in an inflammatory response that was 20
times less intense than the positive control joints. In each case, over
95% of the cells recovered were PMN.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In related work, previous studies by other investigators indicated that in inflammatory arthritis there was evidence of large increases in the concentration of another powerful oxidant, NO (3), and that both the synovial membrane and cartilage were responsible for synthesis of this molecule (19). Further studies by us and others revealed that the activated chondrocytes in the superficial layer of cartilage were responsible for much of the NO produced by this tissue (8, 9). These findings, coupled to previous studies showing the presence of immune complexes trapped on the surface of rheumatoid cartilage (20), suggested that in this disease, large concentrations of oxygen radicals and NO may coexist at the cartilage surface. Finally, recent studies in our laboratory indicated that H2O2, and the product of oxidation of NO, nitrite salts, as well as ONOO, were able to cross-link and nitrosate immunocomplexes (10). The above-mentioned studies raised the question of whether the chemical modification of IgG by these oxidants resulted in an enhancement or a decrease of the well known proinflammatory properties of this molecule. Thus, in the present studies we have systematically investigated the biologic properties of IgG pertinent to the generation of inflammation.
The pertinence of this work was highlighted by the confirmation of previous data indicating that the synovial fluids contained significant amounts of nitrosated and chlorinated IgG species. Previous data using mAbs specific for nitrotyrosine had detected this modified amino acid in whole synovial fluids (21). The present work used the purified IgG obtained from the fluids to ascertain that the nitrosated amino acid was present within this molecule. In the case of chlorinated modification, it was not necessary to isolate the protein because detection was conducted by an independent methodology using specific Abs reacting only against IgG modified by HOCl. The results obtained indicated the presence of higher absolute amounts of the modified IgGs in rheumatoid fluids than in osteoarthritic specimens, although the differences were less distinct when the values were corrected for the IgG concentrations in each fluid. These results reflect levels of the modified proteins at one time point; they do not take into account the rates of formation, catabolism, ingress, and egress from the joint cavity, which may be quite different in inflammatory and noninflammatory arthritides. As a matter of fact, the concentrations of the modified IgGs in each synovial fluid did not correlate with the cell concentrations measured at the time of joint aspiration.
The Fc domain of IgG mediates many of the biological properties of Abs once the Ab site binds to Ag and two or more molecules attached to neighboring epitopes are placed in close proximity to each other. Activation of the complement cascade mediates many of these properties mainly through the action of the C3 convertase of the classical pathway on the third complement, C3. In the present work, we show that brief exposure of the IgG molecule to increasing concentrations of either HOCl or ONOO results in a dose-dependent inhibition of complement activation, as reflected by binding to the modified IgGs of the first component of the cascade, C1q, and C3, the molecule responsible for many of the proinflammatory properties of complement. With small variations, treatment of the IgG with both oxidants at 0.2 mM or greater resulted in a significant decrease in complement component binding. In this respect, Griffiths and Lunec (22) showed that in vitro treatment of IgG with HOCl concentrations as high as 0.2 mM did not decrease C1q binding to that molecule. This apparent discrepancy may be explained by the fact that these investigators used smaller concentrations of HOCl, and also by the very nature of the solid-phase C1q immunoassay used, which did not take into account the degree of aggregation of the IgGs tested. Thus, the treated molecules may have had decreased binding capacity for C1q and increased aggregation simultaneously, leading to the negative results obtained. Moreover, they did show a decrease in C1q binding when the IgG was exposed to activated PMN presumably containing myeloperoxidase, a situation where the cells produce significant amounts of HOCl.
The Fc domain of IgG is also responsible for the mediation of inflammatory cell activation and the secretion of many proinflammatory factors. Oxidative modification of this domain with concomitant inhibition of binding to the membrane receptor and significant decrease in cell activation as demonstrated by H2O2 secretion by PMN would also contribute importantly to a reduction of the severity of the inflammatory process. It should be pointed out that the present experiments were designed to distinguish the effects of the chemical modification of the IgGs from the proinflammatory effects resulting from intermolecular cross-linking and aggregation. In the experiments conducted to gauge IgG binding to the FcR, for instance, it was important to use purified monomers, because the cooperativity effects of two or more binding domains in close proximity may increase the apparent binding affinity by as much as 104.
Are the concentrations of oxidants used in these experiments
physiologically relevant? The concentrations used in our experiments
approach the concentrations of
H2O2 achieved by PMN in
vitro (0.2 mM) (23). Moreover, a significant proportion
of the H2O2 made may be
used to generate HOCl (24), and it is likely that the
concentrations achieved within phagolysosomes are even higher.
Synovial tissue-derived autoantibodies present within the joint cavity in RA are thought to contribute significantly to the chronic inflammatory process (1). Thus, the studies described here involving the effects of the oxidants on the business end of the Ab molecules, the Ag binding domains, suggest that one of the possible beneficial effects of NO and oxygen radicals may reside in their ability to decrease the formation of immunocomplexes. Pertinent to this discussion is the observation that rodents with absent inducible NO synthase developed inflammatory arthritis of similar magnitude to their wild-type counterparts (25, 26), although other pathologic processes such as vasculitis were ameliorated (26).
Finally, the in vitro studies suggesting inhibition of the proinflammatory properties of IgG by the biological oxidants used were confirmed by the in vivo studies in rabbit knees. In these studies, equal amounts of oxidant-modified aggregated IgG injected intraarticularly induced an acute inflammatory response that was significantly less severe than that induced by heat-aggregated IgG of similar molecular mass. Hewitt et al. (27) had previously shown that UV-treated IgG injected into an already inflamed rat air pouch induced worsening of inflammation when compared with the native molecule. It is difficult to compare our results to those of Hewitt et al. We introduced IgG in a normal joint cavity and gauged the results 2 days later, whereas they injected the IgGs into an already inflamed air pouch and found differences 7 days after injection. They used UV light-modified IgG, we used HOCl and ONOO. Moreover, in the present experiments only 100 µg were used per joint, whereas Hewitt et al. injected 5 mg in the air pouches. Although these authors stated that UV treatment did not result in cross-linking, if only 2% of the irradiated IgG became aggregated, it could account for the proinflammatory effects observed.
It is clear that although our results are suggestive, confirmation of the biologic importance of these observations would ideally involve testing the inflammatory capacity of modified IgG aggregates obtained from rheumatoid synovial fluids. Unfortunately, it may be very difficult, if not impossible, to purify enough oxidatively modified aggregates from aggregates generated by other mechanisms, i.e., true immunocomplexes, complexes formed by partial enzymatic digestion of IgG, etc., to compare their inflammatory activities.
Both reactive oxygen species and NO and derivatives have been shown to have protective or proinflammatory effects, depending on the concentrations and the models of inflammation used (1, 7, 28). The studies described here may be pertinent to inflammatory processes in which Ag-autoantibody reactions and immunocomplex pathogenesis play an important role.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Hugo E. Jasin, University of Arkansas for Medical Sciences, Mail Slot 509, 4301 West Markham, Little Rock, AR 72205.
3 Abbreviations used in this paper: HOCl, hypochlorous acid; PMN, polymorphonuclear leukocytes; ONOO, peroxynitrite; H2O2, hydrogen peroxide; RA, rheumatoid arthritis.
Received for publication June 16, 2000. Accepted for publication September 5, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Dhiman, E. S. Nakayasu, Y. H. Madaiah, B. K. Reynolds, J.-j. Wen, I. C. Almeida, and N. J. Garg Enhanced Nitrosative Stress during Trypanosoma cruzi Infection Causes Nitrotyrosine Modification of Host Proteins: Implications in Chagas' Disease Am. J. Pathol., September 1, 2008; 173(3): 728 - 740. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Dimitrov, J. Bayry, S. Siberil, and S. V. Kaveri Sialylated therapeutic IgG: a sweet remedy for inflammatory diseases? Nephrol. Dial. Transplant., May 1, 2007; 22(5): 1301 - 1304. [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kanayama, M. Cascalho, and H. Ohmori Analysis of Marginal Zone B Cell Development in the Mouse with Limited B Cell Diversity: Role of the Antigen Receptor Signals in the Recruitment of B Cells to the Marginal Zone J. Immunol., February 1, 2005; 174(3): 1438 - 1445. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
