Immunocytochemical detection of interleukin 1 within stimulated human monocytes.

We have used synthetic peptides coupled to KLH to raise high titer antisera to human IL-1 beta, and in the present report show the usefulness of these sera for immunocytochemical analyses of IL-1 production. Using indirect immunofluorescence, we have been able to specifically identify IL-1 within human monocytes and to monitor its accumulation with time. After indirect immunofluorescent staining of LPS- and PHA-stimulated mononuclear cell cultures, intense cytoplasmic fluorescence was observed in 93% of the monocytes, but not in lymphocytes or platelets present in the same preparation. Unstimulated monocytes did not contain immunocytochemically detectable IL-1. When put into culture, however, some of the otherwise unstimulated monocytes subsequently showed a transient accumulation of intracellular IL-1. Monocytes cultured in the presence of LPS and PHA exhibited detectable fluorescence after 2.5 h, and the fluorescent intensity of these cells continued to increase over the course of 21 h. Fluorescent staining was abolished by preincubation of the sera with relevant but not irrelevant peptide, and while preimmune or anti-KLH serum produced no staining, antisera against either the amino terminus or an internal region of IL-1 beta produced identical staining patterns. Immunoblot analyses of lysates from stimulated monocytes showed that the antisera against IL-1 recognize a single intracellular species with an apparent molecular weight (33 kD) similar to that predicted for IL-1 precursor from the nucleotide sequence of IL-1 cDNA. The ability to specifically identify and immunocytochemically localize IL-1 within producing cells should prove extremely useful for studying the in situ production of IL-1 in immune-based and inflammatory diseases.

The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.

Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix organization over myotubes and fibroblasts suggest that each of these cell types uses somewhat different means to regulate the assembly of extracellular matrix components within its domain. (c) The limited co-distribution of different components within the extracellular matrix in vitro and the selective immune precipitation of each antigen from conditioned medium suggest that each extracellular matrix component is secreted in a form that is not complexed with other matrix constituents.

Specific bioactivities of monocyte-derived interleukin 1 alpha and interleukin 1 beta are similar to each other on cultured murine thymocytes and on cultured human connective tissue cells.

In this report we compare the bioactivities of pure, human monocyte-derived interleukin 1 (IL-1) alpha and beta in the standard murine thymocyte proliferation assay, a human dermal fibroblast proliferation assay, and in an assay measuring stimulation of prostaglandin E2 (PGE2) release from human rheumatoid synoviocytes. In each case the different species of IL-1 produced saturable stimulation and gave similar dose response curves. Half-maximal stimulation was observed at average IL-1 concentrations of 29 pM in the thymocyte assay, 2 pM in the dermal fibroblast proliferation assay, and 5 pM in the synovial cell assay. Our results show that native, monocyte-derived IL-1 alpha and IL-1 beta are both potent stimulators of connective tissue cells and that the specific bioactivities of these molecules are similar to each other in tests on human connective tissue cells, as well as on murine lymphoid cells.

Identification of a high-affinity receptor for interleukin 1 alpha and interleukin 1 beta on cultured human rheumatoid synovial cells.

In this report the binding of recombinant human interleukins 1 alpha and 1 beta (rIL-1 alpha and rIL-1 beta) to primary cultures of human rheumatoid synovial cells is measured and compared to the concentrations of these mediators required for stimulation of PGE2 production by these same cells. The average concentration of IL-1 alpha required for half-maximal stimulation of PGE2 was 4.6 +/- 1.5 pM (+/- SEM) (n = 6), whereas for IL-1 beta half-maximal stimulation was observed at a concentration of 1.3 +/- 0.24 pM (n = 6). Both direct and competitive binding experiments were performed. In direct binding experiments, IL-1 alpha bound with a Kd of 66 pM (n = 1), while IL-1 beta bound with a Kd of 4 pM (n = 2). In competitive binding experiments, IL-1 alpha inhibited binding of 125I-IL-1 alpha with a Ki of 33-36 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 51-63 pM (n = 2). IL-1 beta inhibited binding of 125I-IL-1 alpha with a Ki of 2-3 pM (n = 2) and binding of 125I-IL-1 beta with a Ki of 7 pM (n = 2). The binding data were best fit by a model specifying a single class of receptors with homogeneous affinity for either IL-1 alpha or IL-1 beta and with an abundance of 3,000-14,000 sites per cell. Autoradiography showed that the vast majority of the synoviocytes within the cultures possessed IL-1 receptors. Comparison of biological response curves with the binding curves indicates that the observed receptors exhibit sufficiently high affinity to mediate the response of human synoviocytes to low picomolar concentrations of IL-1 alpha and IL-1 beta.

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis.

The destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. The role of activated matrix metalloproteinases (MMPs) in the chondrodestructive process was studied by identifying a selective cleavage product of aggrecan in murine arthritis models initiated by immunization with either type II collagen or proteoglycan. We conducted semiquantitative immunocytochemical studies of VDIPEN341 using a monospecific polyclonal antibody requiring the free COOH group of the COOH-terminal Asn for epitope detection. This antibody recognizes the aggrecan G1 domain fragment generated by MMP [i.e., stromelysin (SLN) or gelatinase A] cleavage of aggrecan between Asn341-Phe342 but does not recognize intact aggrecan. VDIPEN was undetectable in normal mouse cartilage but was observed in the articular cartilage (AC) of mice with collagen-induced arthritis 10 d after immunization, without histological damage and clinical symptoms. This aggrecan neoepitope was colocalized with high levels of glycosaminoglycans (GAGs) in pericellular matrices of AC chondrocytes but was not seen at the articular surface at this early time. Digestion of normal (VDIPEN negative) mouse paw cryosections with SLN also produced heavy pericellular VDIPEN labeling. Computer-based image analysis showed that the amount of VDIPEN expression increased dramatically by 20 d (70% of the SLN maximum) and was correlated with GAG depletion. Both infiltration of inflammatory cells into the synovial cavity and early AC erosion were also very prominent at this time. Analysis of adjacent sections showed that both induction of VDIPEN and GAG depletion were strikingly codistributed within sites of articular cartilage damage. Similar results occurred in proteoglycan-induced arthritis, a more progressive and chronic model of inflammatory arthritis. These studies demonstrate for the first time the MMP-dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis.

Multiple forms of (Na+ + K+)-ATPase in the chicken. Selective detection of the major nerve, skeletal muscle, and kidney form by a monoclonal antibody.

Evidence is presented that monoclonal gamma-immunoglobulin secreted by hybridoma-24 recognizes the sodium and potassium ion-stimulated ATPase of neurons, muscle fibers, and kidney tubule cells in the chicken. The antigen consists of alpha (Mr = 105,000) and beta (Mr = 47,000) subunits. Only the beta subunit bears major oligosaccharide additions (including binding sites for wheat germ agglutinin) to its polypeptide core (Mr = 32,000). The detergent-solubilized antigen sediments as a 7 S complex of alpha beta or alpha beta 2. The antigen has a basolateral distribution in the plasma membrane of renal tubule cells, and in myogenic cell cultures there is up-regulation of the antigen in low potassium medium. Monoclonal antibody-24 specifically recognizes purified gull salt gland (Na+ + K+)-ATPase as well as the major candidate molecule for the (Na+ + K+)-ATPase in enriched preparations from chicken kidney. On cultured myotubes, the number of ouabain-binding sites (4.8 X 10(5)/nucleus) and antigenic sites (4.4 X 10(5)) are approximately equal. However, the antigenic sites on fibroblasts (1.1 X 10(4)) account for only about 4% of the ouabain-binding sites, and there is little or no antibody binding to capillary endothelial cells, Schwann cells, or erythrocytes. Evidence is presented that the antigenic determinant is proteinaceous. It is concluded that at least two antigenically distinct (Na+ + K+)-ATPases exist in the chicken.

Characterization of a plasma membrane glycoprotein common to myoblasts, skeletal muscle satellite cells, and glia.

A plasma membrane glycoprotein common to embryonic chick myoblasts and adult chicken skeletal muscle satellite cells is the antigen recognized by monoclonal antibody C3/1. Although traces of the same antigen are present on some muscle-derived fibroblasts, the density of antigenic sites on myoblasts and satellite cells is so high that these cell types can be identified in tissues by immunocytochemical techniques. The antigen is exposed on the surfaces of myogenic cells growing in tissue culture and can be solubilized with detergent. This and other criteria establish that the antigen is a plasma membrane protein. The antigen, purified by affinity techniques, consists of a single type of polypeptide chain which migrates as a relatively broad band of apparent molecular weight 38,000 Da in SDS-polyacrylamide gel electrophoresis. It has a very small sedimentation constant, suggesting that the solubilized form is either monomeric or dimeric. The concentration of antigenic sites increases during myogenesis in vitro; but during maturation the antigenic sites are lost from muscle fibers. Electron microscopic autoradiographic study of adult muscle labeled with iodinated monoclonal antibody demonstrated unequivocally that the antigenic sites in adult muscle are concentrated in the satellite cells. Although selective for myoblasts, immature myotubes and satellite cells in the myogenic lineage, the monoclonal antibody also binds at rather high levels to peripheral Schwann cells and teloglia, to some nonneuronal cells in cultures derived from embryonic spinal cord, to some glial elements of adult chicken brain, and to several cell types in the early embryo.

Extracellular matrix-cytoskeletal interactions in rheumatoid arthritis. I. Immunoelectron microscopic analysis of the fibronexus at the adhesive surface of normal porcine type B synoviocytes in vitro.

We studied cell surface interactions between the fibronectin (FN)-containing extracellular matrix and the actin cytoskeleton of normal porcine synoviocytes in vitro, using electron microscopic methods. These type B synovial cells were distinguishable from dermal fibroblasts co-isolated from the same organism, because of their very long cellular processes and their ability to synthesize prostaglandin E2 after stimulation with interleukin-1. With plastic sections, we found end-to-end (tandem) and track-like (lateral) transmembrane associations of extracellular fibers and cortical 5-nm microfilaments localized along the attenuated synoviocyte processes in postconfluent cultures. Very similar FN-actin complexes, termed fibronexus (FNX), have been observed on cultured fibroblasts and on granulation tissue myofibroblasts in vivo. Using double-label immunoelectron microscopy with monospecific antibodies applied to ultrathin frozen sections of synoviocytes cut in situ, we proved that these FNX were indeed composed of associated FN and actin filaments. The striking finding of numerous FNX in cultured type B synoviocytes strongly suggests that the FNX is a major cell surface adhesion site in normal synovium, which may play an important role in pannus formation, connective tissue remodeling, and synoviocyte proliferation in patients with rheumatoid arthritis.

Analysis of IL-1 and TNF-alpha gene expression in human rheumatoid synoviocytes and normal monocytes by in situ hybridization.

Human IL-1 alpha, IL-1 beta, and TNF-alpha mRNA expression was examined in peripheral blood monocytes (PBM) from normal individuals and in primary synoviocytes isolated from patients with rheumatoid arthritis by Northern blot and in situ hybridization. Cells cultured in the presence or absence of LPS were analyzed using in vitro synthesized 35S-labeled sense or antisense RNA probes to determine the relative abundance and the cell type expressing each of the mRNA for these potent inflammatory mediators. The results indicated that 72% of the LPS-stimulated PBM expressed detectable levels of IL-1 alpha mRNA, 89% IL-1 beta mRNA, and 10% TNF-alpha transcripts. Thus, the majority of activated PBM produced both IL-1 alpha and IL-1 beta. Experiments combining immunofluorescence for IL-1 beta protein with in situ hybridization for TNF-alpha mRNA demonstrated that monocytes expressing TNF-alpha mRNA also produced IL-1 beta. Primary synoviocytes from four patients with RA were also examined for the mRNA expression of each cytokine. Northern blot analyses of total RNA isolated from 0 to 72 h after LPS- or mock-stimulation showed that IL-1 beta mRNA was the most abundantly expressed, followed by TNF-alpha. In situ hybridization revealed that IL-1 beta and TNF-alpha transcripts were detected exclusively in synovial tissue macrophages. IL-1 alpha mRNA was not detected in these cultures by either method.

Evaluation of alveolar macrophages in normals and individuals with active pulmonary sarcoidosis for the spontaneous expression of the interleukin-1 beta gene.

We have evaluated the hypothesis that the presence of large numbers of activated helper/inducer T lymphocytes in the lungs of individuals with active pulmonary sarcoidosis is associated with the exaggerated release of interleukin-1 (IL-1) by alveolar macrophages. Evaluation of media from unstimulated cultured sarcoid alveolar macrophages failed to detect IL-1 activity. When parallel cultures of sarcoid and normal alveolar macrophages were stimulated with lipopolysaccharide (LPS), they released similar amounts of IL-1 activity. Using a highly specific polyclonal anti-IL-1 beta antibody and flow cytometry to evaluate cell-associated IL-1 beta, analysis of fresh alveolar macrophages from patients with active sarcoidosis and normal individuals revealed no detectable cell-associated IL-1 beta, but IL-1 beta was present when macrophages from sarcoid patients and normals were stimulated with LPS. Similar observations were made using immunoblot analysis of cell lysates of the same unstimulated and stimulated macrophages. Finally, Northern analysis of alveolar macrophages for IL-1 beta mRNA transcripts demonstrated minimal, but equivalent, amounts of IL-1 beta in both normal and sarcoid macrophages, as compared to the much larger quantities present in LPS-stimulated alveolar macrophages. Thus, while alveolar macrophages of individuals with sarcoidosis are clearly capable of expressing the IL-1 beta gene, these findings suggest that altered expression of the IL-1 beta gene by alveolar macrophages does not play a central role in the exaggerated lung T-cell activation characteristic of sarcoidosis.

Production, purification and characterization of canine prostromelysin.

One of the best studied animal models of osteoarthritis is a dog model in which the anterior cruciate ligament of the hind limb stifle joint is transected (Pond-Nuki model). To determine whether stromelysin might play a role in this model, it was necessary to purify the enzyme for production of suitable probes. In the present study, dog synovial fibroblasts were stimulated to express a metalloproteinase that was demonstrated to be canine prostromelysin by Northern blot, protein purification and amino-terminal sequence analyses. Unlike rabbit synoviocytes, passaged dog synoviocytes did not express stromelysin mRNA in response to recombinant human IL-1, but expressed stromelysin mRNA only upon treatment with dog monocyte-conditioned medium (dMCM). The aminophenylmercuric acetate (APMA)-activatable metalloproteinase present in the culture supernatants of stimulated dog synoviocytes was purified using a combination of ion-exchange and dye matrix affinity chromatography. The purified canine metalloproteinase co-migrated on reducing SDS-PAGE with recombinant human prostromelysin-1 as a doublet with apparent molecular masses of 54 and 56 kDa. Similar to APMA-activated human prostromelysin-1, the APMA-activated canine metalloproteinase was inhibited by 1,10-phenanthroline or recombinant human tissue inhibitor of metalloproteinase (TIMP). The amino-terminal sequences of the canine pro- and APMA-activated enzymes were compared with those of human, rabbit and rat stromelysin. The striking homologies among the sequences demonstrated that the purified canine metalloproteinase was indeed canine prostromelysin. A rabbit anti-canine prostromelysin polyclonal antiserum was generated and used to localize the enzyme within cultured dog synoviocytes and articular cartilage stimulated with dMCM. The reagents developed in this study should be useful for examining the expression and distribution of prostromelysin in canine models of osteoarthritis.

In vivo expression of stromelysin in synovium and cartilage of rabbits injected intraarticularly with interleukin-1 beta.

OBJECTIVE: To examine the in vivo expression of the matrix metalloproteinase stromelysin in the synovium and articular cartilage of rabbits injected intraarticularly with recombinant human interleukin-1 beta (IL-1). METHODS: The direct isolation of messenger RNA (mRNA) from articular cartilage without the prior isolation of chondrocytes is described. The in vivo expression of stromelysin was examined at the mRNA level by Northern blot analysis, and at the protein level by in situ immunolocalization and by enzyme-linked immunosorbent assay. RESULTS: In the synovium of IL-1-injected joints, stromelysin mRNA levels were highest at 4 hours and declined to background levels within 24 hours. In the cartilage of IL-1-injected joints, stromelysin mRNA was elevated at 4 hours and continued to increase until 8 hours, before declining. Stromelysin mRNA expression preceded a similar increase in stromelysin protein levels in both synovium and cartilage. CONCLUSION: Intraarticular injection of IL-1 induced the endogenous expression of stromelysin mRNA and protein in both synovium and cartilage. The kinetics of stromelysin expression correlated well with the accumulation of stromelysin and proteoglycan in synovial fluids. Therefore, the de novo synthesis of stromelysin in cartilage may have contributed to the loss of proteoglycan from that tissue.

The four biochemically distinct species of human interleukin 1 all exhibit similar biologic activities.

The supernatants of human monocytes incubated with endotoxin are able to stimulate the proliferation of murine thymocytes in the presence of PHA. This is known as LAF (lymphocyte activating factor) activity and is a characteristic activity of interleukin 1 (IL 1). The LAF activity can be resolved into four major fractions: a 15,000 dalton (pI 7), a 15,000 (pI 5.5), a 35,000 (pI 7), and a 35,000 (pI 5.5) fraction. To determine whether these four fractions shared the other biologic activities ascribed to IL 1, they were compared in a series of bioassays. When standardized with respect to their LAF activities, the four fractions did not differ significantly as mitogens for murine thymocytes, inducers of IL 2, murine or human B cell activators, human chondrocyte or synoviocyte stimulants, or inducers of acute phase proteins in vivo. On the other hand, the samples differed markedly as stimulators of porcine synoviocytes, with the 15,000 dalton (pI 5.5) fraction being the only strongly active fraction. These results are consistent with the hypothesis that all four LAF could be products of a single gene, although the porcine receptor may be able to distinguish between them. If this is the case, all four fractions can properly be termed IL 1.

Kinetics of aggrecanase- and metalloproteinase-induced neoepitopes in various stages of cartilage destruction in murine arthritis.

OBJECTIVE: Two major cleavage sites, one mediated by metalloproteinases (MMPs) and the other by an as-yet unidentified enzyme termed aggrecanase, have been observed in aggrecan. To learn more about the relative contribution of these enzymes during cartilage degradation, this study assessed the occurrence of both specific neoepitopes in cartilage during murine arthritis and examined the correlation between neoepitope formation and different aspects of cartilage damage. METHODS: Reversible cartilage damage was induced in mice in the zymosan-induced arthritis (ZIA) model, partly irreversible cartilage damage in the antigen-induced arthritis (AIA) model, and irreversible, destructive cartilage damage in the collagen-induced arthritis (CIA) model. Immunolocalization techniques were used to detect the specific C-terminal neoepitopes VDIPEN (MMPS) and NITEGE (aggrecanase). RESULTS: In normal cartilage from young adult mice, no VDIPEN epitopes were detected, but a limited amount of NITEGE epitopes were already present. During the early phase of proteoglycan (PG) depletion, NITEGE expression was raised substantially in all arthritis models. VDIPEN epitopes were not detected in this early phase of cartilage destruction. When PG depletion progressed toward advanced cartilage damage, VDIPEN epitopes were induced. During ZIA, minimal induction of VDIPEN was observed, whereas in AIA, strong, but partly reversible, VDIPEN staining was evident, and in CIA, an extensive presence and persistence of the MMP-induced neoepitope was seen. When VDIPEN epitopes were intensely present, NITEGE epitopes were greatly reduced at that site in the cartilage. CONCLUSION: Presence of VDIPEN epitopes in cartilage correlated with severe cartilage damage, but these epitopes were not detected during early PG degradation. This suggests a limited role for VDIPEN-inducing MMPs in early PG degradation during murine arthritis. In contrast, aggrecanase epitopes were induced before the appearance of VDIPEN epitopes, but they disappeared with progression of cartilage damage.

Interleukin-1 receptor antagonist prevents expression of the metalloproteinase-generated neoepitope VDIPEN in antigen-induced arthritis.

OBJECTIVE: To investigate the relationship between occurrence of the matrix metalloproteinase-generated neoepitope VDIPEN and proteoglycan (PG) loss in arthritis, and to examine the role of interleukin-1 (IL-1) in VDIPEN expression. METHODS: VDIPEN expression was investigated in murine antigen-induced arthritis by immunolocalization studies on joint sections. The involvement of IL-1 in VDIPEN expression was studied by blocking of IL-1 using IL-1 receptor antagonist (IL-1Ra). RESULTS: Profound PG loss was evident early in arthritis, without significant VDIPEN expression. Full expression of the neoepitope appeared after a few days, when PG depletion was severe, and disappeared at late stages when cartilage showed recovery from PG depletion. At sites where chondrocyte death occurred and cartilage did not recover from the initial cartilage depletion, VDIPEN expression remained present. Prophylactic IL-1Ra treatment of arthritic mice resulted in almost complete prevention of VDIPEN expression. However, IL-1Ra had only a minor effect on PG depletion, emphasizing that there is no correlation between VDIPEN and early PG depletion. CONCLUSION: This study indicates that IL-1 is involved in VDIPEN expression. Although VDIPEN-inducing metalloproteinases do not seem to be involved in early PG depletion during antigen-induced arthritis, metalloproteinase neoepitopes are present when PG depletion is severe.

Induction of increased levels of proteoglycan fragments in synovial fluid (SF) and increased levels of stromelysin in cartilage, synovium and SF by intraarticular injection of canine monocyte conditioned medium into dogs.

OBJECTIVE: To study the effects of the intraarticular injection of canine monocyte conditioned medium (cMCM) into dogs on proteoglycan fragment and stromelysin levels in the joint. METHODS: cMCM was injected intraarticularly into dogs, and the levels of proteoglycan fragments in synovial fluid (SF) as well as stromelysin levels in cartilage, synovium, and SF were assessed after 12 h. RESULTS: There was a 4-fold increase of proteoglycan fragment levels and a 6-fold increase in stromelysin levels in SF, and a 4.4-fold increase in stromelysin levels in cartilage extracts. Elevated mRNA levels were detected in both synovium and cartilage. By immunofluorescence staining, stromelysin was localized in chondrocytes throughout the cartilage and in synovial cells. CONCLUSION: Intraarticular injection of cMCM stimulated the expression of stromelysin mRNA and protein in cartilage and synovium and caused marked increases in stromelysin protein and proteoglycan fragment levels in SF.

Use of an antibody against the matrix metalloproteinase-generated aggrecan neoepitope FVDIPEN-COOH to assess the effects of stromelysin in a rabbit model of cartilage degradation.

OBJECTIVE: To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo. METHODS: The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescence. Proteoglycan fragments in joint fluids were quantified by a dimethylmethylene blue dye-binding assay. RESULTS: Stromelysin cleavage of rabbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN: An anti-FVDIPEN antibody recognized the FMDIPEN neoepitope in situ in cartilage from stromelysin-injected joints. The appearance of the FMDIPEN neoepitope corresponded to the release of cartilage proteoglycan fragments into the joint fluid, and could be inhibited by pretreatment of the rabbits with a synthetic stromelysin inhibitor. CONCLUSION: These results indicate that the anti-FVDIPEN antibody can be used to assess the role of MMPs in cartilage degradation in vivo.