Focus on pain mechanisms and pharmacotherapy in the treatment of fibromyalgia syndrome.

OBJECTIVE: To critically evaluate the role of several notable 'pain pathways' in the fibromyalgia syndrome (FMS). METHODS: PubMed provided the data base for peer-reviewed basic and clinical science studies on musculoskele-tal and neuropathic pain mechanisms with a principal emphasis on critically appraising papers from 2002 to the present. RESULTS: FMS pharmacotherapy is more prevalent in clinical practice as our understanding of the cellular, molecular and pathophysiologic mechanisms contributing to widespread musculoskeletal and neuropathic pain has emerged. Thus, several 'pain pathways' including high-voltage activated Ca2+ channels and the K(v)1 family of K+ channels ion channels appear related to the efficacy of pregabalin and amitryptyline, respectively, in FMS. Additionally, serotonergic and serotonergic/norepinephrine receptor-mediated mechanisms may explain the reported pharmacologic efficacy in FMS of mirtazapine, duloxetine and milnacipran. By contrast, the decreased level of micro-opioid receptors in the CNS of FMS patients suggests a mechanism as to why opioid therapy should be avoided. However, increased peripheral benzodiazepine receptors on monocytes from FMS patients suggested an explanation for the reported efficacy of olanzapine in FMS. CONCLUSION: Pregabalin was the first drug approved by the FDA for the treatment of FMS-related pain. Drugs that have been assessed for their potential use in FMS pharmacotherapy include gabapentin and tricylic antidepressants. These drugs appear to target specific Ca2+ or K+ ion channels notable for their involvement in mediating neuropathic pain. Serotonin and norepinephrine reuptake inhibitors including, mirtazapine, duloxetine and milnacipran appear to be more efficacious in FMS than selective serotonin reuptake inhibitors. Milnacipran became the second FDA-approved drug for FMS.

Small molecular weight inhibitors of stress-activated and mitogen-activated protein kinases.

The stress-activated protein kinase (SAPK) and mitogen-activated protein kinase (MAPK) sub-families are crucial to environmental stress responses and responses to growth factors that cause transcriptional activation of genes required for cell proliferation, differentiation and programmed cell death. Small molecular compounds with specific structure/activity characteristics have been developed that competitively block SAPK/MAPK binding to ATP. Chemically modified compounds based on ATP binding pocket characteristics have improved selectivity and specificity for SAPK/MAPK isoforms. In addition, site-specific mutagenesis of MAPKs has helped identify the MAPK structures required for binding recognition and selectivity of these inhibitors. A group of extracellular-signal regulated protein kinase (ERK) inhibitors has been constructed based almost exclusively on their ability to inhibit the ERK activation cascade. Inhibitors have been employed in vitro to identify protein targets and mechanism of action of SAPKs/MAPKs. The efficacy of SAPK/MAPK inhibitors in animal models of inflammation, arthritis, heart failure, cancer and neurological degeneration has provided the impetus for using them in human studies of inflammation and in clinical trials.

Involvement of caspase-3 in epigallocatechin-3-gallate-mediated apoptosis of human chondrosarcoma cells.

Green tea polyphenol-(-)epigallocatechin-3-gallate (EGCG)-is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis. In this study we describe a novel observation that EGCG displayed strong inhibitory effects on the proliferation and viability of HTB-94 human chondrosarcoma cells in a dose-dependent manner and induced apoptosis. Investigation of the mechanism of EGCG-induced apoptosis revealed that treatment with EGCG resulted in DNA fragmentation, induction of caspase-3/CPP32 activity, and cleavage of the death substrate poly(ADP-ribose)polymerase (PARP). Pretreatment of cells with a synthetic pan-caspase inhibitor (Z-VAD-FMK) and a caspase-3-specific inhibitor (DEVD-CHO) prevented EGCG-induced PARP cleavage. The induction of apoptosis by EGCG via activation of caspase-3/CPP32-like proteases may provide a mechanistic explanation for its antitumor effects.

Tumour necrosis factor alpha enhances the expression of hydroxyl lyase, cytoplasmic antiproteinase-2 and a dual specificity kinase TTK in human chondrocyte-like cells.

Tumour necrosis factor alpha (TNF-alpha) is a cytokine with pleiotropic effects on cells ranging from proliferation to apoptosis. These biological effects of TNF-alpha are believed to be elicited by the induction or enhancement of the expression of TNF-alpha responsive genes in the target cells. TNF-alpha is pro-inflammatory and a principal mediator in the pathogenesis of arthritis. The activation of an inflammatory cascade by TNF-alpha in arthritis results in the degradation of cartilage, joint destruction and loss of function. Because TNF-alpha is an important mediator in the pathogenesis of arthritis, the present study addresses the identification of novel TNF-alpha responsive genes in HTB-94 cell line which is of human origin and maintains a chondrocytic phenotype. The three identified cDNAs were previously not known to be induced or upregulated by TNF-alpha in chondrocytes or cells of chondrocytic lineage. One of the identified cDNAs had sequence similarity to human hydroxyl lyase mRNA (PLOD), an enzyme involved in collagen biosynthesis and its metabolism; the second cDNA had sequence similarity to the human cytoplasmic anti-proteinase-2 mRNA (CAP-2), a member of a group of proteins shown to be associated with protecting cells from TNF-alpha-induced apoptosis; and the third cDNA had sequence similarity to a dual specificity kinase, TTK, which is associated with cell proliferation. Relative gene expression level analysis by PCR and by Northern blotting revealed that treatment with TNF-alpha enhanced the expression of PLOD, CAP2 and TTK transcripts which confirmed the results obtained with display gels. Furthermore, TTK mRNA expression was also induced in human articular chondrocytes treated with TNF-alpha but not in untreated chondrocytes. Our results suggest that these genes may play a role in chondrocytic responses to TNF-alpha-mediated stimuli affecting the cartilage homeostasis.

Metabolic disturbances and synovial joint responses in osteoarthritis.

Previously held views that the pathogenesis of idiopathic osteoarthritis (OA) originated in the synovial joint and was not influenced by systemic metabolic disturbances in the patient is inconsistent with recent data demonstrate skewing of the growth hormone/insulin-like growth factor-1 axis in the symptomatic OA patient. In light of this novel information, the role of growth hormone and insulin-like growth factor-1 in the pathogenesis and progression of OA requires further definition. In male patients with OA, the red blood cell sequesters more growth hormone than an aged-matched control group. Thus, this growth hormone "depot" may provide a mechanism for removal of "toxic" levels of growth hormone from the circulation. Storage of "excess" growth hormone in red cells may reduce the inflammatory or otherwise undesirable "toxic" actions of GH. In some patients, serum growth hormones levels may exceed three-times the average value considered normal. These "episodic" variations in growth hormone levels may play a significant role in the elevated levels of serum growth hormone seen in the OA patient. The connection between elevated growth hormone and decreased insulin-like growth factor-1 levels and the defined cartilage anabolic and catabolic pathways defined in in vitro assays of articular cartilage derived from the OA patients remain to be more precisely defined. However, the dampened insulin-like growth factor-1 response in OA coupled with elevated cartilage extracellular matrix degradation (mediated by metalloproteinases) and depressed compensatory biosynthesis (induced and perpetuated by the presence of cytokines such as interleukin-1 and tumor necrosis factor-alpha) may, in fact, act synergistically to suppress normal cartilage repair mechanisms thus resulting in progressive destructive lesions of the cartilage and bone.

Changes in third carpal bone articular cartilage after synovectomy in normal and inflamed joints.

OBJECTIVE: To determine if arthroscopic synovectomy in normal and inflamed joints had temporal or site-related effects on articular cartilage. STUDY DESIGN: Alterations in equine third carpal bone articular cartilage were studied at two time periods: groups 1 and 2 (6 weeks) and groups 3 and 4 (2 weeks) after synovectomy in normal (groups 2 and 4) and inflamed carpi (groups 1 and 3). ANIMAL POPULATION: 16 carpi from eight horses. METHODS: Biochemical and biomechanical properties of dorsal and palmar articular cartilage were determined by radioloabeling, proteoglycan (PG) extraction, chromatography, electrophoresis, and indentation testing. RESULTS: Synovectomy in inflamed joints produced the greatest concentration of newly synthesized PG in articular cartilage by 2 weeks. Synovectomy in normal joints produced significantly greater newly synthesized PG in articular cartilage by 6 weeks. Dorsal sites had greater newly synthesized and endogenous PG in some groups. Chromatographic profiles of newly synthesized PG demonstrated early and late PG peaks. Electrophoresis of late PG peak showed a toluidine blue-positive band that comigrated with human A1D1 PG monomer in the two groups with the most newly synthesized PG> This band was reactive with monoclonal antibody 1C6 specific for the hyaluronic acid-binding region of aggrecan. For the material properties evaluated, only Poisson's ratio was significantly decreased between groups as a function of time (6 weeks < 2 weeks). and this was most pronounced in the thicker dorsal sites. CONCLUSIONS: Synovectomy in inflamed joints produced site-specific, significantly greater responses in articular cartilage as compared with synovectomy in normal joints. CLINICAL RELEVANCE: Synovectomy may not be beneficial to the articular cartilage in inflamed joints.

Effect of synovial membrane infection in vitro on equine synoviocytes and chondrocytes.

OBJECTIVE: To determine the functional response of synovium to infection, and the influence of infected synovium on articular cartilage metabolism. SAMPLE POPULATION: Synovium and articular cartilage explants from the midcarpal and tarsocrural joints of adult horses. PROCEDURE: For experiment 1, synovium explants were incubated as follows: control--incubation in standard medium, infected (I)--incubation with Staphylococcus aureus, and infected-filtered (IF)--incubation with medium collected from the infected group and filtered (0.22-micron filter). Daily collected medium was assayed for interleukin 1 beta (IL-1 beta), IL-6, tumor necrosis factor, and hyaluronan (HA) concentrations. For experiment 2, cartilage explants were incubated as follows: control--incubation in standard medium, and IF--incubation in medium collected from infected synovium cultures and filtered. After 48 hours, explant proteoglycan synthesis and endogenous proteoglycan and glycosaminoglycan contents were determined. RESULTS: IL-1 beta and IL-6 values were significantly increased in synovium explants from the I and IF groups. Hyaluronan concentration was lower in I and IF groups. Proteoglycan synthesis and content, and total glycosaminoglycan and chondroitin sulfate concentrations, were significantly decreased in cartilage from the IF group. CONCLUSIONS: Bacterial infection was associated with decreased HA concentration and increased mediator release. These effects were also observed despite elimination of bacteria. Exposure to sterile but previously infected medium decreased articular cartilage matrix synthesis and composition. CLINICAL RELEVANCE: Resident synovial cells may contribute appreciably to articular damage during bacterial infection in the absence of migrant inflammatory cells. This response is prolonged despite elimination of the bacteria.

Synthesis of aggrecan core proteins by human cartilage and chondrocytes in vitro.

Proteoglycans synthesized by human cartilage were studied in explants and in chondrocyte cultures. The D1 fraction proteoglycans radiolabelled with 35SO4 were analyzed by 3-16% sodium dodecyl sulfate polyacrylamide gel electrophoresis or on 3-5% acrylamide gels. After 24 h in culture, osteoarthritic and age matched nonarthritic cartilage synthesized one aggrecan core protein (LI) whose apparent size after chondroitinase ABC/keratanase digestion was approximately 520 kDa. Three proteoglycan core proteins were found in the D1 fraction of the medium compartment of nonarthritic chondrocytes, whose apparent sizes were approximately 520 kDa (LI), approximately 390 kDa (LII), and approximately 480 kDa (LIII). The LII and LIII core proteins were routinely absent from osteoarthritic chondrocytes. Chondrocytes from a 28-year-old patient with osteoarthritis secondary to congenital hip dysplasia synthesized principally the LI proteoglycan core protein.

Deficient type I protein kinase A isozyme activity in systemic lupus erythematosus T lymphocytes.

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by a dysfunctional cellular immune response. We have previously identified a metabolic disorder of the adenylate cyclase/cAMP/protein kinase A (AC/cAMP/PKA) pathway characterized by impaired cAMP-inducible, PKA-catalyzed protein phosphorylation in intact T lymphocytes from subjects with severe SLE disease activity. Because this metabolic disorder may contribute to abnormal T cell immune effector functions, we tested the hypothesis that impaired PKA-dependent protein phosphorylation is the result of a PKA isozyme deficiency in SLE T lymphocytes. Compared with healthy and rheumatoid arthritis (RA) controls, subjects with severe SLE activity exhibited reduced PKA-catalyzed phosphorylation of proteins in the T lymphocyte plasma membrane where the type I isozyme of PKA (PKA-I) is predominantly localized. Both silver staining and biosynthetic labeling of membrane-associated proteins with [35S]methionine demonstrated that reduced protein phosphorylation was not due to either an altered distribution of or absence of proteins. Moreover, phosphorylation of SLE membrane-associated proteins with the PKA catalytic (C) subunit showed a similar distribution and extent of phosphorylation compared with membrane proteins from healthy T cells, suggesting that SLE T cell membrane proteins could be phosphorylated. Sequential column chromatography of the type I and type II isozymes of PKA (PKA-I, PKA-II) demonstrated a deficiency of PKA-I isozyme activity. Compared with a ratio of PKA-I to PKA-II activity of 4.2:1 in healthy T cells, the activity ratio in T cells from subjects with severe SLE disease activity was 0.99:1 (P = 0.01, SLE versus healthy controls for PKA-I). The deficient PKA-I activity was associated with a significant increase of free C-subunit activity (P = 0.04, SLE versus healthy controls for C-subunit). T cells from subjects with mild/moderate SLE disease activity also exhibited diminished PKA-I activity, yielding a ratio of PKA-I to PKA-II activity of 2.4:1. By contrast, T cells from RA controls possessed increased PKA-I, PKA-II, and free C-subunit activities compared with healthy controls, resulting in a ratio of PKA-I to PKA-II activity of 3.6:1. We conclude that the reduced PKA-catalyzed protein phosphorylation in the plasma membrane of SLE T cells is the result of deficient PKA-I isozyme activity. This is the first identification of a deficiency of PKA activity in SLE T lymphocytes; the deficiency, resulting in diminished protein phosphorylation, may alter cellular homeostasis, contributing to the cellular immune dysfunctions observed in SLE.

The effect of nonsteroidal anti-inflammatory drugs on cAMP-dependent protein kinase-mediated phosphorylation by human chondrocytes in culture.

Although a principal pharmacological action of nonsteroidal anti-inflammatory drugs (NSAIDs) is to blunt eicosanoid synthesis by inhibiting cyclooxygenase, recent evidence indicates that NSAIDs may also interact directly with proteins that control the activity of adenylyl cyclase. Since the only physiological mechanism governing the action of cyclic AMP occurs via activation of its receptor, cyclic AMP-dependent protein kinase (cAMPPk; Kinase A), we determined whether NSAIDs affect intracellular substrate phosphorylation dependent on cAMPPk. The incorporation of 32Pi into cellular proteins that are substrates for cAMPPk was determined in intact human non-arthritic, aged non-arthritic and osteoarthritic chondrocytes in the presence or absence of NSAIDs, namely, sodium meclofenamate, indomethacin, tiaprofenic acid and sodium salicyclate. The transfer of [32P]-ATP was employed to identify phosphoproteins in a membrane fraction prepared from chondrocyte homogenates in the presence or absence of these NSAIDs. The lowest concentration of NSAID was similar to NSAID concentrations achieved during therapy for the arthritides. In intact human chondrocyte strains, activation of cAMPPk by dibutyryl cAMP (dBcAMP) resulted in the phosphorylation of intracellular substrates with an apparent M(r) of 55kD, 42kD, 26kD, 25kD, 22kD, 21.5kD, 20.5kD, and 17kD when examined by autoradiography after 12.5% SDS/PAGE. The NSAIDs augmented or potentiated phosphorylation of these proteins which were cAMPPk-dependent. In the chondrocyte membrane fraction, protein phosphorylation by cAMP was mimicked by isobutylmethylxanthine or by the purified catalytic subunit of bovine cAMPPk. NSAIDs augmented chondrocyte phosphorylation in the chondrocyte membrane fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Human cartilage from late stage familial osteoarthritis transcribes type II collagen mRNA encoding a cysteine in position 519.

A single base change resulting in the substitution of Cys for Arg at position 519 of the type II collagen triple helix is a predisposing factor in the pathogenesis of a precocious-onset form of familial osteoarthritis associated with a mild chondrodysplasia. Cartilage obtained at the time of total knee replacement in a patient with the Arg-Cys519 mutation was used to investigate the expression of Col2A1 alleles. Using PCR assisted amplification of mRNA with specific amplification of a region of Col2A1 message encompassing exons 31-34, followed by single strand conformation polymorphism and sequence analyses, we have found transcription products of both mutant and normal type II collagen alleles. Further analysis of the sequence of these exons provides evidence that the Arg-Cys519 mutation arose independently in at least two of the three known affected families. The presence of both mutant and normal alleles of Col2A1 in cDNA derived from cartilage obtained from this patient suggests that Cys519-containing type II collagen may continue to be produced even in advanced stages of osteoarthritis.

The effect of circulating serum factors from patients with systemic lupus erythematosus on protein kinase A (PKA) activity and PKA-dependent protein phosphorylation in T lymphocytes.

T lymphocytes from subjects with active systemic lupus erythematosus (SLE) exhibit reduced cAMP-inducible, protein kinase A (PKA)-dependent phosphorylation of several intracellular substrates compared with healthy and disease controls. To ascertain whether the persistent T cell activation observed during active SLE can result in impaired PKA-dependent protein phosphorylation, normal T cells were activated in vitro by monoclonal anti-CD3-epsilon antibody and recombinant IL1-alpha (rIL1-alpha) for 24 hr. T cell activation, verified by IL2 mRNA, IL2 receptor-alpha (IL2R-alpha) mRNA, and IL2R-beta mRNA expression, did not diminish cAMP-inducible, PKA-dependent protein phosphorylation. We also tested the hypothesis that circulating factors present in active SLE serum can decrease cAMP-inducible total PKA phosphotransferase activity and PKA-dependent protein phosphorylation in normal T lymphocytes. T cells cultured for 24 hr in medium supplemented with 10% active SLE sera (from subjects who exhibited the defect of PKA-dependent protein phosphorylation) exhibited similar total PKA phosphotransferase activity and substrate phosphorylation as cells cultured in normal AB serum. Moreover, the addition of interferon-alpha (IFN-alpha) and/or immune complexes (IC) did not diminish either total PKA activity or PKA-dependent substrate phosphorylation. Lastly, we found that the defect of PKA-dependent protein phosphorylation in active SLE T cells could not be reversed by culturing the cells in culture medium supplemented with 10% AB serum for 24 hr. In conclusion, (a) deficient cAMP-inducible, PKA-dependent phosphorylation in SLE T cells is not reversible by culturing cells in vitro; (b) there is no evidence to support the concept that serum factors, including IC and IFN-alpha, can induce a defect of PKA-dependent protein phosphorylation in normal T cells.

Proteoglycan structural changes in human rheumatoid articular cartilage.

Human rheumatoid arthritic (RA) cartilage contains elevated levels of proteolytic enzymes in which the metalloproteases are believed to be the prime enzyme system involved in cartilage metabolism. We examined the effects of these enzymes and the serine proteases on endogenous proteoglycans (PGs) and newly synthesized PGs of seven RA cartilages. The data was further analyzed with regard to the therapy received by the patients prior to surgery. A structural heterogeneity among the PGs from RA cartilage was found, and two subsets were distinguished. While in the first subset more than 35% of the PGs were in aggregate form, no appreciable amount of PG aggregate was found in the second subset. Interestingly, in all but one specimen the subsets appeared to be a function of prior therapy received by the patients. In subset I patients had received prednisone and/or DMARD in addition to NSAIDs, while those from subset II had all received NSAIDs only, with one exception. Our findings also suggest that PG structure alterations could result from the action of already active and APMA-activated metalloproteases; these reduced the PG aggregation capability and caused extensive cleavage in the PG core protein. The serine proteases did not seem to play a major role. Moreover, when the endogenous latent metalloproteases were activated with APMA, high-density PGs (the A1D1 fraction) showed a reduction in their capacity to reaggregate, and in their hydrodynamic size. Using an immunological technique we demonstrated the presence, in subset I, of the hyaluronan binding region domain (HABR) of the core protein. For subset II this domain could not be found.(ABSTRACT TRUNCATED AT 250 WORDS)

Early-onset primary osteoarthritis and mild chondrodysplasia. Radiographic and pathologic studies with an analysis of cartilage proteoglycans.

Three generations of a nonconsanguineous family with premature onset of primary (idiopathic) osteoarthritis (OA) were studied for clues to the etiopathogenesis of their disorder. Articular symptoms began in their second and third decades of life and involved multiple joints, both typical and atypical for primary OA. Radiographs of the majority of involved peripheral joints showed abnormalities typical of primary OA. Evidence of chondrodysplasia was found in the spines. Pathologic examination of femoral heads obtained at total hip arthroplasty from 3 affected family members showed moderate to severe OA. Articular cartilage proteoglycans from these specimens were evaluated for aggregatability with hyaluronic acid, levels of chondroitin sulfate and keratan sulfate, and core protein structure. The results from each patient's specimen differed from the results of the other specimens. We conclude that this family's disorder, primary OA associated with a mild chondrodysplasia, was a late-onset overlap form of an epiphyseal dysplasia, that a defect common to hyaline articular and physeal cartilage was primary, and that a single structural proteoglycan abnormality was not likely to be the underlying cause.

Eicosanoid metabolism by Leishmania donovani-infected macrophages: mouse strain responses in prostanoid synthesis.

Arachidonic acid (20:4) conversion to prostanoids was examined in murine peritoneal macrophages infected in vitro with Leishmania donovani. Four strains of mice differing in resistance to in vivo L. donovani infection were studied. Normal macrophages from all strains converted 20:4 to prostanoids and this was augmented by L. donovani infection. Although cells from each strain synthesized elevated levels of prostaglandin-E2 (PGE2), there were differences with respect to relative increases of this product, with infected macrophages from the C3H/HeJ strain showing the smallest increase above basal levels. These results indicate that macrophages from mouse strains with distinct levels of in vivo resistance to L. donovani all respond to infection with augmented prostanoid synthesis. Although some heterogeneity in strain-specific PGE2 responses to in vitro infection were observed (with respect to increases in PGE2 synthesis above basal levels), it is unlikely that differential resistance of these strains to in vivo infection is strictly related to these relative differences. It seems likely that other genetically controlled factors may have a major impact on disease expression.

Stimulation of sulfated-proteoglycan synthesis by forskolin in monolayer cultures of rabbit articular chondrocytes.

Forskolin, a plant cardiotonic diterpene, stimulated proteoglycan biosynthesis by chondrocytes in monolayer culture. The quantitative increase in proteoglycans was dependent on the concentration of forskolin, but was relatively independent of the presence of serum. At forskolin concentrations that stimulated proteoglycan synthesis, a significant stimulation of adenylate cyclase and cAMP was also measured. The quantitative increase in proteoglycans was characterized, qualitatively, by an increased deposition of newly synthesized proteoglycan in the cell-associated fraction. An analysis of the most dense proteoglycans (fraction dA1) in the cell-associated fraction showed that more of the proteoglycans eluted in the void volume of a Sepharose CL-2B column, indicating that an increased amount of proteoglycan aggregate was synthesized in forskolin-treated cultures. The proteoglycan monomer dA1D1 secreted into the culture medium of forskolin-stimulated cultures overlapped in hydrodynamic size with that of control cultures, although cultures stimulated with forskolin and phosphodiesterase inhibitors produced even larger proteoglycans. The hydrodynamic size of 35SO4 and 3H-glucosamine-labelled glycosaminoglycans isolated from the dA1D1 fraction of the culture medium was greater in forskolin-treated chondrocytes, especially from those in which phosphodiesterase inhibitors had been added. These results indicated that forskolin, a direct activator of chondrocyte adenylate cyclase mimicked the effects of cAMP analogues on chondrocyte proteoglycan synthesis previously reported. These results implicate activation of adenylate cyclase as a regulatory event in the biosynthesis of cartilage proteoglycans, and more specifically in the production of hydrodynamically larger glycosaminoglycans.

Murine monoclonal antibodies recognizing rabbit proteoglycans.

Alterations in the structure and composition of sulfated proteoglycans are found in aging and osteoarthritic rabbits. Monoclonal antibodies (mAB) recognizing specific epitopes of rabbit cartilage proteoglycans would be useful in documenting proteoglycan changes during pathophysiological responses resulting in osteoarthritic pathology in rabbit synovial joints after partial medial meniscectomy. To this point, Balb/c mice were immunized with rabbit proteoglycan (fraction A1D1D1) extracted from xiphoid process. Murine spleen cells were used to prepare hybridomas by fusion with the tumor cell line SP 2/0-Ag 14. Nine mAbs were found to bind to A1D1D1 in a solid phase radioimmunoassay. Binding curves, utilizing A1D1D1 as ligand, resulted in the assignment of mAbs to 3 classes - high, moderate and poor binding mAbs. Binding avidity was independent of immunoglobulin subclass. A1D1D1 was digested with trypsin, chromatographed on DEAE-cellulose and tryptic peptides further resolved by dissociative CsCl density gradient centrifugation. The mAbs were studied in detail utilizing competitive inhibition assays of the resolved peptide fragments. Three types of antigenic fine specificity were observed; a mAb (2G2) which recognized a recurrent epitope on the native A1D1D1, a mAb (2E9) which recognized a single protein epitope, in that it bound to a tryptic peptide that contained a high gluNH2:galNH2 and a mAb (6C9) which preferentially recognized a recurring epitope on heat-treated (50 degrees C minutes) A1D1D1. In this analysis, the epitopes of these mAbs appear to be associated with the core protein since only one mAb (2C7) was competitively inhibited from binding to native A1D1D1 by glycosaminoglycans, hyaluronic acid and oligosaccharides of hyaluronic acid. Direct immunofluorescence staining of rabbit hip, shoulder and knee cartilage showed a differential staining pattern of extracellular matrix with the various mAbs. FITC-2G2 stained the interterritorial matrix intensely; and also the perilacunae zones, whereas FITC-2E9 and FITC-6C9 appeared restricted to the perilacunae regions.