Stimulation of collagenase secretion from rheumatoid synovial tissue by human collagen peptides.

Dayer et al. demonstrated that mononuclear cells from human peripheral blood produce a mononuclear-cell factor that stimulates collagenase secretion from adherent rheumatoid synovial cells. The production of this mononuclear-cell factor can be stimulated by phytohemagglutinin, a T-cell mitogen. We have examined immune mechanisms by which collagenase secretion may be stimulated from explants of rheumatoid synovial tissue and from primary monolayer cultures of rheumatoid synovial cells. Conditioned media from cultures of normal peripheral-blood mononuclear cells that had been activated by phytohemagglutinin stimulated collagenase secretion from most explants and from all monolayer cultures that were examined. The direct addition of phytohemagglutinin stimulated collagenase secretion in explants from eight of fourteen patients and in monolayer cultures from three of six patients. These observations indicate the presence of responsive T lymphocytes in rheumatoid synovial tissue and implicate them in the stimulation of collagenase secretion from synovial cells. The direct addition of type-II collagen peptides to rheumatoid explants also stimulated collagenase secretion in explants from six to fifteen patients. Four of five of these patients displayed moderate or severe disease activity. Other patients showed a marginal stimulation of collagenase secretion on addition of type-II collagen peptides (four of fifteen patients) and type-I collagen peptides (three of twelve patients). None of the patients with mild or inactive disease responded to type-II collagen. With one exception, the addition of collagen peptides to monolayer cultures did not stimulate collagenase secretion. An increased production of factors that enhance or inhibit the migration of polymorphonuclear leukocytes was also observed in explant cultures in response to the addition of phytohemagglutinin (in seven of twelve patients), of type-I collagen peptides (in five of ten patients), and of type-II collagen peptides (in five of eleven patients). There was no correlation between production of these factors and stimulation of collagenase secretion in rheumatoid synovial tissue.

Cross-linking studies on the conformation and dimerization of myelin basic protein in solution.

Myelin basic protein was isolated from both cat and bovine central nervous system. Cat and bovine myelin basic protein, which are shown to be similar by tryptic mapping, exhibit identical behavior when cross-linked with the bifunctional reagent difluorodinitrobenzene. Myelin basic protein is cross-linked into only a dimer under certain conditions in the presence of sodium dodecyl sulfate. In contrast, many oligomers are formed when myelin basic protein is cross-linked in the absence of detergent. The formation of cross-linked dimers in the absence of other oligomer formation suggests that the protein is at least partly dimeric in the presence of sodium dodecyl sulfate. The conformation of them myelin basic protein monomer in sodium dodecyl sulfate was also studied. N-Bromosuccinimide and cyanogen bromide cleavage reactions were used to demonstrate that difluorodinitrobenzene had introduced intramolecular cross-links between the two peptides resulting from each of the cleavage ractions. However, these types of intramolecular cross-links cannot be detected under conditions in which only dimers have formed. Some of the lysine residues which are modified by difluorodinitrobenzene were identified by tryptic mapping. In several respects, the conformation of myelin basic protein in a sodium dodecyl sulfate solution appears to be similar to the conformation of the protein in the membrane.

Protein associations and basic protein conformation in the myelin membrane. The use of difluorodinitrobenzene as a cross-linking reagent.

The near-neighbor relationships of proteins in the myelin membrane were examined using dinitrodifluorobenzene and other cross-linking reagents. When intact cat dorsal column or isolated myelin fragments were treated with cross-linking reagents, up to 20% of the myelin basic protein dimerized. The only other cross-linked product formed in the intact cat dorsal column was a heterodimer consisting of myelin basic protein and either the major or minor proteolipid protein. The remaining myelin proteins, including the major proteolipid protein, were cross-linked into very high molecular weight aggregates. In contrast, when the myelin membrane was dipersed in sodium dodecyl sulfate before the addition of cross-linking reagent, all the proteins remained essentially monomeric, with the exception of myelin basic protein which dimerized to some extent. In the absence of cross-linking reagent, it was shown by radioimmunoassay that small amounts of myelin basic protein dimer and the heterodimer were normally present in sodium dodecyl sulfate-polyacrylamide gels. We found no evidence of intramolecular cross-links between the two peptides formed by N-bromosuccinimide cleavage or between the two peptides formed by cyanogen bromide cleavage of the basic protein monomer. The regions of the myelin basic protein molecule involved in dimerization were also determined by similar cleavage of the cross-linked dimer. A rudimentary model for the structure of basic protein dimer in myelin is presented.

Human synovial cells secrete a 39 kDa protein similar to a bovine mammary protein expressed during the non-lactating period.

By SDS/PAGE analysis we have observed that human synovial cell monolayers secrete a prominent 39 kDa protein which could not be detected in skin and lung fibroblasts. This protein was purified to homogeneity by heparin-Sepharose chromatography and reverse-phase h.p.l.c. The N-terminal sequence was found to be almost identical to that of a recently described bovine protein detected in the mammary secretions during the involutionary phase of the lactational cycle. Characterization of this 39 kDa protein may provide a useful marker for classification of connective tissue cells.

Induction of synthesis and release of interleukin-8 from human articular chondrocytes and cartilage explants.

OBJECTIVE: The activation of neutrophils in the joint space may contribute to the destruction of cartilage matrix observed in rheumatoid arthritis. The capacity of articular chondrocytes to synthesize and secrete interleukin-8 (IL-8) and GRO alpha, two potent neutrophil chemoattractant peptides, was investigated to determine whether cartilage itself could serve as a source of these small cytokines. METHODS: Induction of IL-8 and GRO protein was studied both at the messenger RNA (mRNA) and the protein level by reverse transcriptase/polymerase chain reaction and metabolic labeling, respectively. RESULTS: Strong induction of IL-8 was observed in primary cultures of articular chondrocytes as well as in cartilage explants stimulated with IL-1 beta. The increased secretion of the IL-8 protein was accompanied by corresponding increases in mRNA levels. In contrast to other connective tissue cells, a peptide corresponding in molecular size to the GRO proteins was only weakly induced in cartilage explants or primary chondrocyte cultures. However, mRNA for all 3 members of the GRO family was easily detectable in cultured chondrocytes following stimulation with IL-1 beta. In explanted cartilage, mRNA for only GRO gamma was found to be induced. Newly synthesized IL-8 was slowly released from cartilage explants over a prolonged time in culture. CONCLUSION: The results suggest that synthesis and secretion of the diverse members of the IL-8/GRO family is regulated in a tissue-specific or cell-specific manner. The slow release of IL-8 from articular cartilage following induction by IL-1 beta could establish a chemotactic gradient toward the articular surface and mediate the migration and attachment of neutrophils and lymphocytes to this tissue.

Connective tissue antigens stimulate collagenase production in arthritic diseases.

Peripheral blood mononuclear cells from patients with rheumatoid arthritis (n = 27), systemic lupus erythematosus (n = 24), juvenile rheumatoid arthritis (n = 30), osteoarthritis (n = 20), apparently healthy adults (n = 12), and nonarthritic children (n = 8) were exposed to several putative connective tissue antigens to determine if the monokine, mononuclear cell factor, was released. Release of this factor was detected by bioassay in which enhancement of collagenase production from human synovial cells or dermal fibroblasts was measured. The antigens, all of homologous tissue origin, included cyanogen bromide-derived peptides of type I, II, and III collagens, type I and II helical collagens, and cartilage proteoglycan. Of the subjects examined, 44% of the rheumatoid group, 42% of the systemic lupus group, 33% of the juvenile rheumatoid group but only 10% of the osteoarthritic group and 5% of the control group released monokine after exposure of peripheral blood mononuclear cells to at least one of these connective tissue antigens. Patients with rheumatoid arthritis most frequently responded to type II peptides (but not to type II helical collagen) although the frequencies of responses to type I peptides, type I helical collagen and proteoglycan were also elevated over levels observed in the control population. Positive responses in these patients typically occurred to only one antigen, were transient, often occurred close to the onset of arthritis, and appeared to be unrelated to disease activity. The profiles of responses in patients with juvenile rheumatoid arthritis and systemic lupus shared many features in common and were distinct from those of adult rheumatoid arthritis. Patients with systemic lupus or juvenile rheumatoid arthritis responded to all of the antigens tested. Positive responses often occurred simultaneously to several antigens. Responses to type II helical collagen were most common while sensitization to type II peptides was infrequently detected. Positive responses were transient, unrelated to overall disease activity, type of juvenile arthritis, or duration of disease in lupus patients. Stimulation of mononuclear cell factor release by connective tissue molecules and their degradation products may make an important contribution to the chronic inflammation commonly seen in these diseases.

Cytokine-induced expression of mRNAs for chemotactic factors in human synovial cells and fibroblasts.

In response to interleukin 1 or tumor necrosis factor, human synovial cells and fibroblasts expressed several genes encoding known chemotactic factors or related proteins. Transcripts for interleukin 8 (IL-8), gro/MGSA, pAT 464, IP-10, pAT 744 and Monocyte Chemotactic and Activating Factor (MCAF) accumulated rapidly in IL-1 and TNF-treated cells. The inhibition of protein synthesis led to the superinduction of IL-8 and gro/MGSA mRNAs in IL-1, but not in TNF-treated cells. Thus, IL-1 and TNF are likely to regulate the expression of these mRNAs by different mechanisms. Important cell-specific differences in mRNA accumulation characterized the expression of chemotactic factor genes. Moreover, only a subset of the same genes was activated in quiescent cells stimulated by serum. Therefore, genes encoding closely related proteins each had a distinct pattern of expression. continuous stimulation of fibroblasts and synovial cells with IL-1 resulted in high and prolonged expression of IL-8 and gro/MGSA mRNAs. These results extend the list of chemotactic factor genes expressed by mesenchymal cells in vitro and suggest a pivotal role for these cells in processes such as chronic inflammation.

Organization of membrane proteins in the intact myelin sheath. Pyridoxal phosphate and salicylaldehyde as probes of myelin structure.

Pyridoxal phosphate and salicylaldehyde were used as protein-labeling probes to study the organization of membrane proteins in the intact myelin sheath of the cat dorsal column. Both reagents react with protein amino groups to form Schiff's bases which can be reduced with NaB3H4. The relatively membrane-impermeant pyridoxal phosphate labels all proteins of the intact myelin except basic protein. This major protein of myelin is labeled only after loss of membrane integrity. The relatively membrane-permeant probe, salicylaldehyde, was then used to establish that the basic protein is truly located on the cytoplasmic side of the myelin bilayer, and not merely sequestered within the multiple lamellar structure of the sheath. All proteins in the intact myelin are readily labeled by this reagent, with the label distribution pattern identical to that of disrupted myelin fragments. These data suggest a model for myelin structure in which the basic protein is the only major protein component located exclusively on the cytoplasmic side of the membrane (the major period zone of the sheath), with the other major proteins disposed wholly, or in part, in the extracellular half of the membrane bilayer (the intraperiod zone). All proteins, although asymmetrically disposed with respect to membrane sidedness, appear to be randomly distributed throughout the lamellae which comprise the sheath.

Inflammatory cytokines induce synthesis and secretion of gro protein and a neutrophil chemotactic factor but not beta 2-microglobulin in human synovial cells and fibroblasts.

Exposure of human synovial cells and fibroblasts in monolayer culture to interleukin 1 results in prominent secretion of proteins with Mr values of 6000 and 7000. By N-terminal sequence analysis, the Mr-6000 protein is identified as the protein encoded by a recently described gro mRNA. The Mr-7000 protein is identical to a neutrophil chemotactic factor released from monocytes. Stimulation of normal human fibroblasts with tumour necrosis factor alpha also results in expression and secretion of these two proteins. In addition to these cytokine-induced proteins, we have identified beta 2-microglobulin as an Mr-8000 protein constitutively secreted by synovial cells.

Differential release of plasminogen activator and latent collagenase from mononuclear cell-stimulated synovial cells.

Independent studies have previously shown that mononuclear cell supernatants stimulate the release of plasminogen activator and latent collagenase from synovial cell monolayer cultures. Simultaneous secretion of these enzymes could be an important pathway for tissue destruction under inflammatory conditions, since plasminogen activator can cause activation of latent collagenase in the presence of plasminogen. We investigated the kinetics of release of the two enzymes from synovial cells in response to the addition of mononuclear cell supernatants and retinoic acid. Synovial cells derived from osteoarthritic and rheumatoid arthritic patients responded similarly. Plasminogen activator is released within a few hours of stimulation, and secretion usually stops when the stimulus is removed. In contrast, significant amounts of collagenase are secreted only after an initial lag period of 1--2 days, and secretion is sustained long after removal of mononuclear cell supernatant. Another difference in regulation of the secretion of these two neutral proteinases is that the addition of all-trans retinoic acid to the same synovial cell culture elevates plasminogen activator secretion while suppressing that of latent collagenase. Differential regulation of these enzymes under conditions of chronic inflammation may allow for continual accumulation of latent enzyme(s) which are activated during short periods of plasminogen activator release.

Immune regulation of collagenase secretion in rheumatoid and osteoarthritic synovial cell cultures.

Primary cultures of synovial cells were obtained by proteolytic dispersion of synovial tissue from patients with rheumatoid arthritis (n = 19), psoriatic arthritis (n = 2), osteoarthritis (n = 13) and other joint problems (n = 3). The levels of endogenously secreted collagenase were variable from patient to patient but did not differ significantly between rheumatoid arthritis and osteoarthritis. The endogenous collagenase secretion was likely a consequence of mononuclear cell factor (MCF) release from monocytes/macrophages which have been shown to be present among the heterogeneous primary rheumatoid synovial cell population (Dayer et al., 1976). As also demonstrated by these investigators, medium containing MCF can be generated from peripheral blood mononuclear cells in a T lymphocyte-dependent process by the addition of phytohemagglutinin (PHA). The addition of such medium stimulated collagenase secretion from all our synovial cell cultures regardless of the endogenous level. Protein synthesis but not synovial cell proliferation was required for MCF stimulation of collagenase secretion. The direct addition of PHA to primary synovial cell cultures stimulated collagenase secretion in some but not all cases indicating the presence of T lymphocytes in these positively-responding cultures. In some of these primary synovial cell cultures in which the addition of PHA stimulated collagenase secretion, secretion was also stimulated by the addition of collagen peptides, native collagen, proteoglycan or purified protein derivative of tuberculin. We propose that, in these instances, MCF release is mediated by antigen-sensitized lymphocytes. Antigen-responsive cultures were not restricted to the rheumatoid population. Our data are compatible with the idea that infiltrated lymphocytes in inflamed synovial tissue become sensitized to cartilage and joint capsule components released during tissue degradation and contribute to matrix destruction by mediating MCF release with consequent stimulation of collagenase synthesis and secretion from synovial cells.

Variation in the effects of mononuclear cell products from different individuals on metalloproteinase secretion from human articular cartilage.

Human articular cartilage in organ culture shows a variable degree of endogenous metalloproteinase secretion depending on the individual from whom it was obtained. Cartilage with low endogenous levels are stimulated by interleukin 1 (IL-1) to levels comparable to the high endogenous group. Total blood mononuclear cell products obtained from different individuals either behave in a manner similar to that seen with IL-1, which results in sustained high levels of enzyme secretion, or show an initial stimulation followed by a subsequent dropoff in enzyme secretion even though incubation is continued in the presence of mononuclear cell products. The factor(s) causing this dropoff can be distinguished from IL-1. Production and regulation of such factors may reflect a mechanism whereby the action of IL-1 can be controlled during the inflammatory response.

Mononuclear cell factors stimulate the concomitant secretion of distinct latent proteoglycan, gelatin and collagen degrading enzymes from human skin fibroblasts and synovial cells.

Human skin fibroblasts or synovial cells exposed to conditioned medium from human peripheral blood mononuclear cells release distinct latent enzymes capable of degrading collagen, proteoglycan (PG) and gelatin at neutral pH. The PG-degrading activity also degrades casein. These enzymes require calcium, are activated by 4-aminophenylmercuric acetate and can be inhibited by ethylenediamine tetraacetic acid, but not by phenylmethylsulfonyl fluoride, iodoacetamide or pepstatin. Simultaneous secretion of these proteinases after exposure to conditioned medium from peripheral blood mononuclear cells may be an important mechanism by which connective tissue extracellular matrix is destroyed during chronic inflammation in diseases such as rheumatoid arthritis.

The role of mononuclear cell products as modulators of cartilage degradation.

Human articular cartilage cultured in the presence of interleukin 1 (IL-1) produced from activated peripheral blood mononuclear cells, exhibits the concomitant release of a latent metalloproteinase and fragments of proteoglycan. The manner in which the activated form of the metalloproteinase degrades proteoglycan subunit and link protein suggests a role for this enzyme in cartilage degradation in vivo. While cartilage cultured in the continuous presence of purified IL-1 shows sustained release of metalloproteinase, tissue cultured in the presence of the total products released by activated mononuclear cells from some individuals showed initial stimulation followed by a decline, suggesting the presence of a modulator of IL-1 action.

Human articular cartilage secretes characteristic metal dependent proteinases upon stimulation by mononuclear cell factor.

Human articular cartilage stimulated during organ culture by the addition of human blood mononuclear cell factor concomitantly released latent proteinases and proteoglycan. Gel chromatography at pH 7.4 separated 2 distinct latent enzymes: a general proteinase which could degrade proteoglycan, casein and gelatin, and a collagenolytic activity of smaller molecular size. The general proteinase was calcium dependent and could be activated by either trypsin or 4-aminophenylmercuric acetate; the former procedure invariably generating greatest activity. The enzyme exhibited a broad bimodal pH profile against proteoglycan, with optimum activity at pH 5.5.

Lymphocyte transformation to connective tissue antigens in adult and juvenile rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus, and a nonarthritic control population.

Transformation of peripheral blood lymphocytes after exposure to connective tissue antigens was measured in patients with adult (n = 35) and juvenile rheumatoid arthritis (n = 34), osteoarthritis (n = 21), ankylosing spondylitis (n = 15), and systemic lupus erythematosus (n = 26) and in control subjects (n = 36). The connective tissue antigens included homologous cartilage-type proteoglycan, cyanogen bromide-derived peptides of type I, II, and III collagens, and type I and II helical collagens. Lymphocyte transformation was not detected in the osteoarthritic and control groups, with one exception. Sensitization to at least one connective tissue antigen was detected in approximately one-third of the rheumatoid arthritic and lupus patients and in one-quarter of the juvenile rheumatoid patients. In ankylosing spondylitis, positive responses occurred to proteoglycan in 20% of patients tested but never to collagens or peptides. Sensitivity to proteoglycan was detected only in ankylosing spondylitis except for one patient with juvenile rheumatoid arthritis. In patients with systemic lupus erythematosus and both forms of rheumatoid arthritis, lymphocyte transformation was usually more frequently detected to peptides than to the helical collagens. In adult rheumatoid arthritis, type II peptides elicited an elevated number of responses (14%) as did type I (9%) and III (8%) peptides to lesser degrees. Responses to type I (4%) and II (4%) helical collagens were infrequent. Rheumatoid arthritic patients usually exhibited sensitivity to only one antigen and lymphocyte transformation was often detected when the arthritis was improving. In juvenile rheumatoid arthritis, lymphocyte transformation was detected to peptides of type I (16%), II (9%), and III (29%) collagens and to helical type I (12%) and II (8%) collagens. In systemic lupus erythematosus, sensitization was detected to peptides of type I (13%), II (20%), and III (14%) collagens and to helical type I collagen (18%) but not type II collagen. Simultaneous sensitivity to several antigens often occurred in both systemic lupus erythematosus and juvenile rheumatoid arthritis. Examination of individual patients in all three rheumatic disease groups revealed that immune sensitivity developed to collagen peptides rather than to the helical molecules, particularly in the case of type II collagen. Thus, some patients with inflammatory arthritis exhibit immune responses to connective tissue components which are, as a group, characteristic for each type of arthritis. These responses, which were not obviously associated with disease activity, may develop as a result of inflammation or trauma which destroys connective tissue and exposes molecules, in either a native or degraded state, to cells of the immune system. Expression of sensitivity to these tissue antigens may contribute to the chronicity of the inflammatory arthritides.

Activation of latent collagenase by serum proteinases that interact with immobilized immunoglobulin G.

It has been previously observed that collagen destruction occurs in the vicinity of immune complexes present in articular cartilages of patients with rheumatoid arthritis. When IgG is covalently linked to Sepharose it behaves as if it has reacted with an antigen to form an immune complex, in that it binds the complement component C1 from human serum. Other serum components also interact with this matrix, though their interaction may not be specific for IgG. Two of these components were shown to possess proteolytic activity, one being kallikrein and the other having the properties of plasmin. Both of the activities could activate latent human collagenase. Whilst the binding of the plasmin activity is probably nonspecific, the binding of the kallikrein activity may be selective for IgG (although it is not certain whether this binding is direct or indirect via another molecule). These results therefore suggest that active proteinases such as plasma kallikrein may be selectively concentrated on immune complexes in vivo, where they may locally activate latent proteinases such as collagenase thereby initiating tissue destruction.