Celecoxib versus diclofenac in the management of osteoarthritis of the knee.

OBJECTIVE: A clinical trial was conducted in 600 patients with OA of the knee to test the hypothesis that the specific COX-2 inhibitor, celecoxib, has equivalent efficacy and a superior tolerability/safety profile when compared to diclofenac, the current worldwide standard of care. METHODS: Patients were administered celecoxib 100 mg BID, diclofenac 50 mg TID or placebo for 6 weeks in a multicentre, double-blind. placebo-controlled trial. RESULTS: Primary efficacy measures (index joint pain by VAS, WOMAC index) indicated statistically significant improvement versus placebo for both celecoxib and diclofenac and no statistically significant differences between celecoxib and diclofenac. American Pain Society (APS) measures to assess the rapidity of onset of action showed statistically significant and comparable pain relief versus placebo within 24 h for both celecoxib and diclofenac. More diclofenac patients reported GI side effects than patients treated with either placebo or celecoxib. Diclofenac-treated patients experienced statistically significant elevations in mean hepatic transaminases and serum creatinine and reductions in haemoglobin concentration when compared to placebo, events not observed with celecoxib. CONCLUSION: Celecoxib 200 mg daily is as effective as diclofenac 150 mg daily for relieving signs and symptoms of OA of the knee, including pain, and has a rapid onset of action. However, celecoxib appears to have a superior safety and tolerability profile.

Comparative efficacy and safety of celecoxib and naproxen in the treatment of osteoarthritis of the hip.

Osteoarthritis (OA) is responsible for more disability of the lower extremities in the elderly than any other disease in the US. The pain associated with OA is the primary symptom leading to disability in these patients. Current ACR guidelines recommend consideration of acetaminophen for mild-to-moderate pain and conventional non-steroidal anti-inflammatory drugs (NSAIDs) or COX-2 specific inhibitors for moderate-to-severe OA symptoms. The aim of this study was to compare the efficacy and safety of the COX-1 sparing, COX-2 specific inhibitor, celecoxib, with the conventional NSAID naproxen, and placebo, in the treatment of OA of the hip. In this multicenter, randomized, placebo-controlled trial, 1061 patients with symptomatic OA of the hip were randomized to receive celecoxib at doses of 100 mg, 200 mg, or 400 mg/day; naproxen 1000 mg/day; or placebo, for 12 weeks. Patients were evaluated using standard measures of efficacy at baseline, 2-4 days after discontinuing previous NSAID or analgesic therapy, and after 2, 6, and 12 weeks of treatment. All doses of celecoxib and naproxen significantly improved the symptoms of OA, at all time points compared with placebo. This sustained treatment effect of celecoxib was dose dependent. In terms of pain relief and improvement in functional capacity, celecoxib 200 mg/day and 400 mg/day were similarly efficacious and were comparable to naproxen. Both drugs were generally well tolerated. Celecoxib at a dose of 200 mg/day is as effective as a standard therapeutic dose of the conventional NSAID, naproxen, in reducing the pain associated with OA of the hip.

Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: A randomized controlled trial. Celecoxib Long-term Arthritis Safety Study.

CONTEXT: Conventional nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with a spectrum of toxic effects, notably gastrointestinal (GI) effects, because of inhibition of cyclooxygenase (COX)-1. Whether COX-2-specific inhibitors are associated with fewer clinical GI toxic effects is unknown. OBJECTIVE: To determine whether celecoxib, a COX-2-specific inhibitor, is associated with a lower incidence of significant upper GI toxic effects and other adverse effects compared with conventional NSAIDs. DESIGN: The Celecoxib Long-term Arthritis Safety Study (CLASS), a double-blind, randomized controlled trial conducted from September 1998 to March 2000. SETTING: Three hundred eighty-six clinical sites in the United States and Canada. PARTICIPANTS: A total of 8059 patients (>/=18 years old) with osteoarthritis (OA) or rheumatoid arthritis (RA) were enrolled in the study, and 7968 received at least 1 dose of study drug. A total of 4573 patients (57%) received treatment for 6 months. INTERVENTIONS: Patients were randomly assigned to receive celecoxib, 400 mg twice per day (2 and 4 times the maximum RA and OA dosages, respectively; n = 3987); ibuprofen, 800 mg 3 times per day (n = 1985); or diclofenac, 75 mg twice per day (n = 1996). Aspirin use for cardiovascular prophylaxis (

Cyclooxygenase-2 specificity and its clinical implications.

Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce pain and inflammation by inhibiting the synthesis of prostanoids. However, these drugs inhibit both cyclooxygenase-1 (COX-1), which is essential for the regulation of homeostasis in many tissues, as well as COX-2, which is an important mediator of pain and inflammation. Disruption of COX-1 enzymatic activity by NSAIDs leads to such side effects as interference with platelet functions and gastric ulcers. The recent development of COX-2-specific inhibitors, such as celecoxib, raises the possibility of relieving pain and inflammation with reduced risk of gastrointestinal complications. In Phase II and III studies, celecoxib has demonstrated efficacy in alleviating dental pain and the signs and symptoms of osteoarthritis and rheumatoid arthritis. This COX-2-specific inhibitor was also associated with a markedly lower rate of gastroduodenal injury than is seen typically with NSAIDs. Incidence of most adverse events (including gastrointestinal) and withdrawal rates resulting from adverse events with celecoxib were similar to placebo. Celecoxib appears to be both safe and effective in the treatment of osteoarthritis and rheumatoid arthritis. Its COX-2-specific inhibitory properties thus introduce the possibility of effective relief of arthritic and other types of pain and inflammation with less risk of the mechanism-based toxicities observed with conventional NSAIDs.

Economic and gastrointestinal safety comparisons of etodolac, nabumetone, and oxaprozin from insurance claims data from patients with arthritis.

This study was conducted to compare the effect of etodolac, nabumetone, and oxaprozin use on gastrointestinal (GI) safety and associated costs based on insurance claims information from practice settings. Data were obtained from a national claims database (MarketScan) for the years 1992 to 1994. The claims data of interest were for patients with arthritis who had used etodolac, nabumetone, or oxaprozin exclusively during a 9-month follow-up period (ONLY groups), or these drugs plus (PLUS groups) the other nonsteroidal anti-inflammatory drugs (NSAIDs) ibuprofen, naproxen, diclofenac, sulindac, piroxicam, ketoprofen, or indomethacin. For each group, we obtained information on the use of inpatient and outpatient services for GI-related events and the associated costs. All GI admissions were classified as NSAID-induced or possibly NSAID-induced events based on International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9 CM) codes. All outpatient upper GI ulcers or bleeding episodes were also identified by specific ICD-9 CM code. There were no significant between-group demographic differences. The proportions of patients with NSAID-induced and possibly NSAID-induced GI admissions were 0.1% and 0.4% for the etodolac-ONLY, 0.3% and 1.0% for the nabumetone-ONLY, and 0.1% and 0.5% for the oxaprozin-ONLY groups, respectively (P > 0.05), and a similar pattern was observed among the PLUS groups. In outpatient settings, 3.9%, 4.2%, and 4.9% of the etodolac-, nabumetone-, and oxaprozin-ONLY patients, respectively (P > 0.05), and 6.0%, 5.3%, and 4.7% of the etodolac-, nabumetone-, and oxaprozin-PLUS patients, respectively, had at least one upper GI ulcer/bleeding claim (P > 0.05). The total health care costs for 9 months were approximately $3000 each for the etodolac-, nabumetone-, and oxaprozin-ONLY groups. Oxaprozin, nabumetone, and etodolac had similar GI-safety and associated-costs profiles based on information from practice settings. Also, in patients who used multiple NSAIDs, the groups did not differ in their GI-safety and cost profiles.

Leukocyte migration in immune complex glomerulonephritis: role of adhesion receptors.

The application of our evolving knowledge of adhesion receptors to experimental models of immune complex glomerulonephritis has led to substantial advances in our understanding of how leukocytes emigrate from the vasculature into glomeruli and produce glomerular dysfunction. With respect to neutrophil (PMN) migration and activation in the context of nephritis, the adhesion molecules alpha M beta 2, alpha IIb beta 3, and intercellular adhesion molecule-1 (ICAM-1) seem to be most essential, with more modest (and less well defined) contributions by P-selectin, alpha 4 beta 1, and vascular cell adhesion molecule-1 (VCAM-1). The influx of PMNs is driven largely by complement and alpha chemokines. In contrast to PMNs, monocyte/macrophage migration and activation during nephritis appear to be largely mediated by the adhesion molecules alpha L beta 2, alpha 4 beta 1, ICAM-1, VCAM-1, and potentially P-selectin. Monocyte/macrophage migration also differs from that of PMNs in that it is complement-independent and involves beta chemokines. Further refinement of our understanding of the role of adhesion receptors in immune glomerulonephritis may eventually lead to clinically applicable strategies to ameliorate glomerular inflammation and resulting glomerulosclerosis.

Chemokines are expressed in a myeloid cell-dependent fashion and mediate distinct functions in immune complex glomerulonephritis in rat.

Using anti-glomerular basement membrane nephritis in rats, we investigated the mechanisms underlying in situ chemokine expression and the in vivo function of these cytokines during the acute phase of this model. We observed that CXC chemokine expression was monophasic and paralleled neutrophil (PMN) influx, whereas CC chemokine expression was biphasic with peaks coinciding with the influx of PMNs and macrophages (Mphi). The initial peak of chemokine expression was attenuated by decomplementation, neutropenia, and leukopenia, while the latter peak was attenuated only by leukopenia and augmented in the accelerated form of this disease model, corresponding to an increase in Mphi influx. Differential expression of chemokines by PMNs and Mphi was not an intrinsic property of these cells, as these leukocytes expressed similar profiles of chemokines in vitro. Immunostaining for Mphi inflammatory protein-1alpha, a CC chemokine, in acute nephritis validated that expression during acute nephritis was accompanied by local protein production. Moreover, neutralizing Ab to Mphi inflammatory protein-1alpha attenuated the acute phase proteinuria, but not the accompanying influx of PMNs. Neutralizing Ab to cytokine-induced neutrophil chemoattractant (a CXC chemokine), in comparison, inhibited both PMN influx and proteinuria. A combination of both Abs was not significantly more effective than either alone. In sum, the influx of myeloid cells is necessary for local chemokine expression in anti-glomerular basement membrane nephritis, although the differential expression of CXC and CC chemokines must involve additional factors. CXC and CC chemokines also mediate distinct, but overlapping, pathophysiologic roles in the acute phase of this model.

Macrophage arachidonate release via both the cytosolic Ca(2+)-dependent and -independent phospholipases is necessary for cell spreading.

We have observed that phospholipase A2 (PLA2) activation and arachidonate (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA2S in these processes. The PLA2-specific inhibitors, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL, specific for the Ca(2+)-independent PLA2 (iPLA2)) and methyl arachidonoyl fluorophosphonate (MAFP, specific for the Ca(2+)-dependent phospholipase (cPLA2)) inhibited AA release and cell spreading in a correlated fashion but only modestly decreased cell adherence. Cell spreading was normalized by the addition of AA to PLA2-inhibited cells. AA release during spreading was also inhibited by Ca2+ depletion or protein kinase C (PKC) inhibition, and was accompanied by increased (but transient) phosphorylation of cPLA2-Inhibition of macrophage spreading, however, only partially inhibited AA release. Moreover, constitutive AA release was seen in fully spread macrophages which was inhibited by BEL, but not MAFP or Ca2+ depletion. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the iPLA2 (which appears to be constitutively active) and cPLA2 (which appears to be stimulated by adherence/spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA2.

Polyunsaturated fatty acids and renal disease.

An upregulation of arachidonate metabolism often accompanies renal pathophysiologic states. The resulting eicosanoids contribute to, or modify, the underlying process. Recent investigations suggest that platelet-neutrophil interactions, as well as alterations in the expression of the inducible isoform of cyclooxygenase, play a critical role in mediating changes in arachidonate metabolism in renal inflammation. The importance of arachidonate to renal pathophysiology has been highlighted by prior investigations which have demonstrated a beneficial effect of dietary polyunsaturated fatty acid (PUFA) modulation in a variety of models of experimental renal disease. More recent work has established that this beneficial effect may depend upon alterations in both lipid mediator generation as well as changes in cell function. In light of the benefits of dietary PUFA modulation in models of experimental renal disease, there have been numerous recent clinical trials of dietary (n-3) PUFA supplementation in patients with a variety of renal disorders. These clinical trials suggest that such therapy may be an important addition to the clinical armamentarium, especially in the treatment of IgA nephropathy.

Heterogeneity and clinical significance of glomerular-binding antibodies in systemic lupus erythematosus.

We used an ELISA employing extracts of human glomerular basement membrane (GBM) to detect, characterize, and evaluate the clinical significance of glomerular-binding IgG in patients with SLE nephritis. Most patients with SLE nephritis exhibited GBM-binding IgG, although many patients with active nonrenal SLE or symptomatic, drug-induced lupus had similar reactivity, albeit at lower levels. IgG binding to GBM in SLE nephritis patients was decreased by DNase pretreatment of GBM, restored after DNase with nuclear antigens (most notably with nucleosomes), inhibited by exogenous nuclear antigens (particularly nucleosomes), but unaffected by exposure of serum to DNase/high ionic strength. The characteristics of IgG binding to GBM largely paralleled the patients' underlying autoimmune response, which was dominated either by antibodies to DNA/nucleosomes or to nucleosomes alone. Binding of lupus sera to nonrenal extracellular matrix (even with nucleosomes) was not equivalent to GBM. Collagenase pretreatment of GBM variably decreased IgG binding, depending on the level and type of binding. SLE nephritis patients with high levels of GBM-binding IgG exhibited more severe disease clinically, but the same renal histopathology, as patients with lower levels. The level of GBM-binding IgG at presentation did not predict the therapeutic response, but decreased in responders to therapy. In sum, glomerular-binding IgG in lupus nephritis binds to epitopes on chromatin, which adheres to GBM in part via collagen. These autoantibodies appear necessary, but not sufficient, for the development of nephritis, and correlate with clinical rather than histopathologic parameters of disease activity.

Differing roles of CD18 and VLA-4 in leukocyte migration/activation during anti-GBM nephritis.

The mechanisms underlying leukocyte migration into inflamed glomeruli and their in situ activation are incompletely understood. We addressed this issue by investigating the effects of monoclonal antibodies (mAbs) to CD18 and VLA-4 on these process in the heterologous phase of anti-glomerular basement membrane (GBM) nephritis in rat. Anti-CD18 mAb substantially attenuated neutrophil (PMN) migration into glomeruli and the accompanying proteinuria which is a function of in situ leukocyte activation (ca. 60%). Anti-VLA-4 mAb modestly inhibited PMN migration (ca. 20%) and had no significant effect on proteinuria. Combination of both mAbs was no more effective than anti-CD18 mAb alone. Despite continued mAb blockade of CD18 or VLA-4 (or both), macrophage (M phi) migration following PMN influx was unaltered. However, combined CD18/VLA-4 mAbs diminished the proteinuria associated with M phi influx (ca. 50%). Abrogation of the acute influx of PMNs in this model (via complement depletion or anti-PMN antibody) did not diminish the following influx of M phi s, although the associated proteinuria was abolished. In this context, M phi migration was substantially decreased by anti-VLA-4 mAb (ca. 50%), but not anti-CD18 mAb (either alone or with anti-VLA-4 mAb). In sum, leukocyte migration and activation in the heterologous phase of anti-GBM nephritis are dependent on CD18 and VLA-4, although to varying degrees depending on the leukocyte subtype and the presence of prior inflammation. Nonetheless, a significant component of both PMN and M phi migration/activation is CD18/VLA-4 independent.

Murine glomerulotropic monoclonal antibodies are highly oligoclonal and exhibit distinctive molecular features.

We recently produced a panel of seven glomerular-binding mAbs from a nephritic MRL-lpr mouse that bind to histones/nucleosomes (group I) or DNA (group II) adherent to glomerular basement membrane. To elucidate the molecular basis of their binding and ontogeny, we sequenced their variable (V) regions, analyzed the apparent somatic mutations, and predicted their three-dimensional structures. There were two clonally related sets (3 of 4 in group I, 3 of 3 in group II) both of the VHJ1558 family, and one mAb of the VH 7183 family. V region somatic mutations within clonally related sets had little effect on glomerular binding and did not appear to be selected for based on glomerular binding. The VH regions were most homologous with those from autoantibodies to histones, DNA, or IgG (i.e., rheumatoid factors), the Vkappa regions, with those from autoantibodies to small nuclear ribonucleoproteins (snRNP). The VH regions also exhibited an unusual VD junction (in the group I clonally related set) and an overall high content of charged amino acids (arginine, aspartic acid) in complementarity-determining regions (CDRs), particularly in CDR3. Molecular modeling studies suggested that the Fv regions of these mAbs converge to form a flat, open surface with a net positive charge. The CDR arginines in group I mAbs; appear to be located in Ag contact regions of the binding cleft. In sum, these data suggest that glomerulotropic mAbs are a highly restricted set of Abs with distinctive molecular features that may mediate their binding to glomeruli.

Nephritogenic autoantibodies in lupus: current concepts and continuing controversies.

In summary, we suggest that the following statements regarding lupus nephritis are best supported by the existing data. 1) Lupus nephritis is an immunologically complex disorder. Autoantibodies directed against multiple epitopes on chromatin, including but not limited to dsDNA, may contribute to nephritis. 2) The presence of charged residues within autoantibody heavy chain CDR regions, particularly CDR3, may be essential to the property of nephritogenicity. 3) Chromatin/antichromatin immune complexes (formed either in the circulation or in situ in the GBM) are likely the proximal cause of lupus nephritis. Cross-reactive autoantibodies or antibodies reacting directly to glomerular antigens are less likely to play a major pathogenic role. 4) The induction of lupus nephritis may relate to the propensity of chromatin or its components to bind to the GBM by virtue of the interactions of histones with type IV collagen and heparan-sulfated glycosaminoglycans. Nonetheless, as indicated above, there are numerous issues that remain to be addressed and clarified with respect to lupus nephritis. Insight into these issues is not only of theoretical interest, but may lead to new approaches to diagnostic testing and more specific therapies to replace currently use nonspecific immunosuppressive drugs, which have substantial toxicities.

Modulation of renal disease in autoimmune NZB/NZW mice by immunization with bacterial DNA.

Preautoimmune New Zealand Black/White (NZB/NZW) mice immunized with Escherichia coli (EC) double standard (ds) DNA produce antibodies that bind mammalian dsDNA and display specificities similar to spontaneous lupus anti-DNA. Since calf thymus (CT) dsDNA fails to induce these antibodies, these results suggest a special potency of foreign DNA in inducing serological manifestations of lupus in a susceptible host. To assess the effects of DNA immunization on clinical manifestations in NZB/NZW mice, we measured renal disease and survival of mice immunized with either (a) EC dsDNA as complexes with methylated bovine serum albumin (mBSA) in adjuvant; (b) CT dsDNA with mBSA in adjuvant; (c)mBSA alone in adjuvant; or (d) unimmunized. After immunization with EC dsDNA, NZB/NZW mice developed significant levels of anti-dsDNA antibodies. Nevertheless, these mice had less proteinuria, nitrate/nitrite excretion, and glomerular pathology than mice immunized with either mBSA alone, CT dsDNA/mBSA complexes, or unimmunized mice. Survival of the EC dsDNA immunized mice was significantly increased compared with the other mice. Furthermore, immunization of mice after the onset of anti-DNA production and proteinuria stabilized nephritis and prolonged survival. The improvement in renal disease occurred despite the expression of autoantibodies that bound mammalian dsDNA as well as glomerular antigens. These results suggest that bacterial DNA has immunological properties that attenuate murine lupus despite the induction of pathogenic antibodies.

Murine lupus glomerulotropic monoclonal antibodies exhibit differing specificities but bind via a common mechanism.

We recently identified that the glomerular binding activity in MRL/lpr serum consists of Abs reactive with DNA/histone adherent to glomerular basement membrane (GBM) via type IV collagen. These studies suggest the presence of multiple nephritogenic autoantibodies that bind to glomeruli via a common mechanism, an hypothesis we tested by producing glomerular binding mAbs from a nephritic MRL/lpr mouse. All 7 mAbs produced bound to glomeruli/GBM in a DNase-inhibitable fashion. The 4 mAbs that bound most avidly to glomeruli/GBM (group I) did not bind to DNA per se, and GBM binding after DNase treatment was reconstituted by histones or histone/DNA co-addition. The remaining 3 mAbs (group II) bound well to DNA, and GBM binding after DNase treatment was reconstituted by DNA but not histones. Collagenase (but not heparitinase) inhibited GBM binding of all mAbs and impaired the ability of nuclear Ags to reconstitute binding. None of the mAbs bound to type IV collagen per se. Using defined nuclear Ags, two group I mAbs bound specifically to histone H2A-H2B-DNA complexes, one bound specifically to intact chromatin, and one was polyreactive to histones. Group II mAbs bound to any nuclear Ag-containing DNA. Binding to nuclear Ags by all mAbs was altered if type IV collagen was used as the assay substrate, and in this context the common Ag for all mAbs was intact chromatin. In sum, glomerular binding mAbs exhibit differing antigenic specificities, but can be identified as either predominantly anti-histone/nucleosome (group I) or anti-DNA (group II). Regardless, binding to chromatin adherent to type IV collagen is a common property of all such mAbs.

GRO chemokines: a transduction, integration, and amplification mechanism in acute renal inflammation.

We recently observed that cytokine-induced neutrophil chemoattractant (CINC), a GRO chemokine, contributes to neutrophil migration into the inflamed glomerulus in rat. Therefore, we sought to clarify how expression of the GRO chemokines, CINC and macrophage inflammatory protein-2 (MIP-2), is regulated in mesangial cells in vitro and the kidney in vivo. Mesangial cells expressed both GRO chemokine mRNAs in response to mediators of acute renal inflammation [interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharides (LPS)], but not chronic renal inflammation (transforming growth factor-beta 1), with CINC mRNA expression predominating over MIP-2. The kinetics of GRO chemokine mRNA expression in response to both IL-1 beta and TNF-alpha (but not LPS) paralleled those defined for polymorphonuclear leukocyte (PMN) migration during nephritis in vivo. IL-1 beta and TNF-alpha displayed nonparallel concentration-response relationships for GRO chemokine mRNA expression, and together were synergistic together rather than additive. Expression of GRO chemokine mRNAs in response to both cytokine agonists, however, was inhibited by genistein, a tyrosine kinase inhibitor. GRO chemokine mRNAs were rapidly expressed in inflamed glomeruli during immune complex glomerulonephritis with MIP-2 predominating over CINC. Expression of both chemokines was substantially inhibited by complement, leukocyte, and PMN depletion. In sum, GRO chemokines are expressed coordinately by mesangial cells and inflamed glomeruli and appear both to transduce the response to mediators of acute inflammation into a chemotactic signal and to amplify this response both temporally and quantitatively.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of variable-region somatic mutations on the specificity and pathogenicity of murine monoclonal anti-DNA antibodies.

Antibodies to DNA (anti-DNA) occur prominently in systemic lupus erythematosus and provoke inflammatory damage in the kidneys. To determine the factors that confer pathogenicity on antibodies of this specificity, we investigated the in vitro and in vivo glomerular binding by members of four clonally related sets of monoclonal anti-DNA antibodies from lupus mice. Somatic mutations within the clonal sets enhanced binding to double-stranded DNA (dsDNA). Binding to permeabilized glomeruli in vitro was observed among affinity-purified preparations of these antibodies independent of specificity for dsDNA. In normal mice injected with hybridoma cell lines, nephritis as assessed by histology and immunofluorescence did not correlate with antibody affinity for DNA. By multivariate analysis, in vitro glomerular binding was the most predictive parameter of histologic outcome. These findings indicate that somatic mutations occurring during maturation of the autoimmune response do not necessarily enhance pathogenicity.

Discordance between macrophage arachidonate metabolic phenotype and the expression of cytosolic phospholipase A2 and cyclooxygenase.

Macrophages (M phi s) exhibit variations in their ability to release and metabolize arachidonate (AA) depending on their state of activation, differentiation, and tissue origin. In order to understand these variations on a molecular level, we determined whether differences in AA release and metabolism by murine peritoneal M phi s could be explained in terms of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX) expression. Resident M phi s exhibited greater COX capacity (conversion of exogenous AA to PGE2) but lower phospholipase (PLase) activity (release of endogenous AA) than elicited M phi s. Activation of resident M phi s in vivo with endotoxin increased both their PLase activity and COX capacity. Despite the observed differences in PLase activity, peritoneal M phi s under all conditions expressed similar amounts of cPLA2 mRNA and protein. All M phi s exhibited COX-1 mRNA and protein (i.e., the constitutive isoform of COX), although elicited M phi s exhibited increased mRNA for COX-1 but decreased levels of protein, relative to resident M phi s. Elicited (but not resident) cells also exhibited COX-2 mRNA but not COX-2 protein (i.e., the inducible form of COX). Despite the increased COX capacity of resident cells with in vivo activation, their expression of COX-2 mRNA and protein was equivalent to that of unactivated cells, becoming apparent only after cell adherence in vitro. In sum, there is no simple relationship between the ability of M phi s to release and metabolize AA, and the expression of cPLA2 or COX isoforms. Moreover, adherence appears to be important for the expression of COX-2 by M phi s.

Glomerular binding activity in MRL lpr serum consists of antibodies that bind to a DNA/histone/type IV collagen complex.

To clarify the pathogenesis of lupus nephritis, we developed an assay that defines a glomerular binding activity (GBA) in both murine and human lupus sera highly correlated with nephritis. In the current study, we used a cross-adsorption strategy to establish that the GBA in MRL lpr serum binds to the glomerular basement membrane (GBM). We subsequently observed that this binding to the GBM was competitively inhibited by either exogenous DNA or histone, abrogated by pretreatment of the GBM with DNAse, and restored after DNase treatment with DNA/histone in a synergistic fashion. GBM binding was also completely inhibited by pretreatment of GBM with collagenase but not heparatinase. The effect of collagenase was not reversed by the subsequent addition of DNA, but was restored by the sequential re-addition of type IV collagen and DNA. By using purified basement membrane components, we found that MRL lpr serum bound avidly to DNA coated on type I collagen but less well (or not at all) to DNA coated on type IV collagen, laminin, or fibronectin. Histone pretreatment of type IV collagen before DNA addition, however, synergistically enhanced binding in a fashion similar to that seen with native GBM. Thus, the GBA in MRL lpr serum seems to be comprised of autoantibodies that bind to histones and/or DNA that adhere to type IV collagen within the GBM. These data support the planted Ag hypothesis as the principal pathogenic mechanism in lupus nephritis and suggest that multiple autoantibodies may contribute to this disorder.

Acute inflammation is the harbinger of glomerulosclerosis in anti-glomerular basement membrane nephritis.

The degree of glomerular inflammation and injury during immune-mediated glomerulonephritis is felt to be critical to the eventual development of glomerulosclerosis, although the relative contributions of acute and chronic inflammation are uncertain. By grading the initial dose of antibody in accelerated anti-glomerular basement membrane nephritis, we observed that only animals with the most substantial acute inflammation (in terms of neutrophil influx and acute proteinuria) developed sustained proteinuria followed by an increase in serum creatinine and evidence of severe glomerulosclerosis. Chronic inflammation (i.e., glomerular macrophage influx and evidence of glomerular cell proliferation), in contrast, was evident without the development of glomerulosclerosis. Decreasing the degree of acute inflammation during severe nephritis by complement depletion diminished both the initial and sustained proteinuria and the influx of neutrophils, prevented the terminal increase in serum creatinine, and attenuated the evolution of glomerulosclerosis. Complement depletion, however, did not affect peak proteinuria, macrophage influx, or glomerular cell proliferation. Regression analysis of the entire data set demonstrated that acute (day 1) proteinuria was predictive of the eventual histopathological index, more so than chronic (day 7) proteinuria. To recapitulate, glomerulosclerosis following antiglomerular basement membrane nephritis appears to be substantially dependent on the degree of acute inflammatory injury.