SYNOVIAL CELL ACTIVATION INDUCED BY A POLYPEPTIDE MEDIATOR *

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73993/1/j.1749-6632.1975.tb36057.x.pd

Connective Tissue Activation. II. Abnormalities of Cultured Rheumatoid Synovial Cells

Rheumatoid synovial cells grown in vitro demonstrated: a) increased rate of lactate formation, b) increased rate of glucose uptake, c) increased rate of hyaluronate synthesis, d) decreased sensitivity to exogenous activator and e) increased concentrations of an endogenous activator. Exogenous activator was shown to be capable of inducing cortisol unresponsiveness in relation to suppression of hyaluronate synthesis. Evidence that exogenous activator is not long retained by synovial cells suggests that elevated levels in rheumatoid cells are due to accentuated endogenous formation. Elevated activator peptide content of rheumatoid synovial cells provides an explanation for many of the differences between normal and rheumatoid synovial cells in vitro.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37717/1/1780140108_ftp.pd

Connective tissue activation

Connective tissue a ctivating p eptides from lymphocytes (CTAP-I) and platelets (CTAP-III) are known to stimulate glycosaminoglycan synthesis, glycolysis, and mitogenesis in connective tissue cell cultures. Direct evidence suggested that increased accumulation of cyclic AMP was involved in the action of these peptide agonists, and increased prostaglandin E synthesis was postulated on the basis of indirect evidence. In the present experiments, CTAP-I and -III were incubated with human and murine cells in culture, and prostaglandin E was measured by radioimmunoassay using antibody directed primarily to prostaglandin E2. Both CTAP-I and -III markedly stimulated the elaboration of prostaglandin E into culture medium, the earliest evidence of increased synthesis occurring at 4 hours with maximal concentrations found at 24 hours. Substantial residual stimulation persisted at least through 48 hours. Indo-methacin (13.0 Mg/ml) obliterated basal and incremental synthesis of prostaglandin in the presence of mediators. Cycloheximide (8.7 Mg/ml) did not affect the stimulation of prostaglandin synthesis by CTAP-I and -III. Three nonrheumatoid and 3 rheumatoid synovial cell strains showed similar basal levels of prostaglandin E and similar responses to CTAP-I. A murine fibroblast cell strain (3T3) showed increased prostaglandin E synthesis on exposure to CTAP-I, and the KB tumor cell strain was markedly stimulated by CTAP-III. These studies confirm the increased synthesis of E series prostaglandins postulated to occur in human connective tissue cells on exposure to CTAP-I and -III, and clarify the mechanism of action of these agonists on “activated” target cells. The importance of elevated extracellular concentrations of prostaglandins is uncertain, although they may act directly on sensitive cell types as well as potentiate the actions of CTAP-I and -III on neighboring cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37752/1/1780240309_ftp.pd

Leukocyte-connective tissue cell interaction. II. The specificity, duration, and mechanism of interaction effects

Human fibroblasts grown in vitro as monolayer cultures were “activated” by syngeneic lymphocytes, polymorphonuclear leukocytes, and fibroblasts as well as by allogeneic leukocytes, human embryonic kidney cells, fibroblasts, and thrombocytes. “Activation” was characterized by increased medium acidity, increased glucose uptake, increased lactate formation, and marked stimulation of hyaluronic acid formation. The “activation” process was blocked by preincubation with actinomycin D, but not by incubation with 2,4 dinitrophenol. “Activated” fibroblasts continued to exhibit increased mucopolysaccharide synthesis for 5 to 28 days following a single exposure to stimulatory materials.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37712/1/1780120405_ftp.pd

Connective Tissue Activation. I. The Nature, Specificity, Measurement and Distribution of Connective Tissue Activating Peptide

A polypeptide which induces hypermetabolism in normal synovial fibroblasts in tissue culture is described. The metabolic changes induced included a marked increase in hyaluronate formation, lactate formation and glucose uptake; formation of soluble and fibrous collagen was depressed. An assay method is presented which permits quantitative comparisons of samples for connective tissue activating peptide (CTAP) activity. The CTAP appeared to be widely distributed in human tissues, the amount of activity being a function of cell density. A role for CTAP in rheumatoid inflammation is proposed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37716/1/1780140107_ftp.pd

Identification of acid mucopolysaccharides by paper chromatography

A multiple solvent paper chromatography system is described which tentatively identifies most of the known mammalian acid mucopolysaccharides, when these are examined singly. Certain mucopolysaccharides may be identified with reasonable certainty in complex mixtures of such substances. Protein contamination of significant degree does not appear to interfere with these methods. These chromatographic systems appear to be most useful for the rapid, tentative identification of acid mucopolysaccharides where only limited samples are available.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32161/1/0000216.pd

Connective tissue activation

Human synovial fibroblasts in culture have been stimulated to augment hyaluronate synthesis and glucose utilization by connective tissue activating peptides (CTAP) extracted from human spleen, lymphocytes, platelets, granulocytes, and tumor cells. The platelet-derived mediator CTAP-III also stimulated DNA synthesis in synovial fibroblasts, but CTAP-I from lymphocytes and spleen did not. The present study demonstrates the mitogenic potential of a granulocyte mediator (CTAP-PMN). Normal granuiocytes were prepared with Ficoll-diatrizoate gradients, platelet contamination being estimated by phase microscopy and by radioimmunoassay for the platelet-specific protein, Β thromboglobulin. CTAP-PMN preparations derived from 4 × 10 7 cells/ml stimulated culture 3 H-thymidine incoporation to 3.56 ± 1.32 (SD) times controls levels. Although exposure of praparations to thiols reduced their mitogenicity, CTAP-PMN was relatively heat-stable. SDS gel eletrophoresis of active fractions suggested a molecular weight between 12,700 and 12, 700 daltons. In doulbe immunodiffusion, antisera to CTAP-III showed no reactivity with CTAP-PMN. CTAP-PMN or other granulocyte factors capable of stimulating fibroblast DNA synthesis may play a role in chronic proliferative synovitis or in other settings where exudative inflammation is accompanied by connective tissue growth.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37746/1/1780230506_ftp.pd

Review of united states data on neoplasms in rheumatoid arthritis

Relatively sparse literature developed during the past 30 years that sought to characterize the relationship of rheumatoid arthritis to neoplasms. The past decade has seen added concern over possible oncogenic effects of cytotoxic agents now used to manage some patients with rheumatoid arthritis. Acquisition of unambiguous data is complicated by the fact that the cumulative incidence of cancer in the general population exceeds 30 percent, and that most studies have insufficient patient numbers, duration follow-up, and attention to age, sex, race, or known etiologic agents. Thus, it is not surprising to find reports that cancer incidence is high, low, or unchanged in rheumatoid arthritis. Although equally ambiguous data were accumulated concerning potential neoplasm-inducing effects of cytotoxic drugs, concern is justified in relation to increased frequency of bladder cancer after cyclophosphamide and acute leukemia following alkylating agents.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25802/1/0000365.pd

A method for measuring acid mucopolysaccharides in serum-containing tissue culture media

The details of a simple, relatively rapid method for measuring uronic acid-containing acid mucopolysaccharides in protein-rich tissue culture media are presented. The procedure would appear to be most useful in those situations where hyaluronic acid and chondroitin sulfate are the primary mucopolysaccharides present.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32374/1/0000449.pd

An improved method for immobilizing IgG antibodies on protein A-agarose

This report describes a modification of a procedure developed by others for crosslinking IgG to protein A which itself is covalently linked to a gel support. Earlier immunoaffinity columns were described as having large antigen-binding capacities and stability under a variety of elution conditions. The present data show that columns constructed with earlier techniques were only partially stable to pH 3.0 buffers, and, as a result, bound less than 20% of the antigen predicted by theory. Modifying parameters of the dimethylpimelimidate crosslinking method led to immunoaffinity columns which did not leak immunoglobulin under low pH elution buffer conditions. The new immunoaffinity absorbants, because of the increased strength of the couple between the antibody and protein A, were capable of binding antigen at over 80% of their theoretical capacity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28682/1/0000499.pd