Hugo E. Jasin

Suppressor Function of Peripheral Blood Mononuclear Cells in Normal Individuals and in Patients with Systemic Lupus Erythematosus

Normal peripheral blood mononuclear cells demonstrated increased DNA synthesis and secretion of newly synthesized protein when suboptimal concentrations of Concanavalin A (Con A) were added to the cultures after 24-h incubation in vitro. Cells stimulated by Con A, 1 mug/ml, after 24-h incubation demonstrated 3.0 times more tritiated thymidine incorporation, and 4.4 times more 14C-amino acid incorporation into newly synthesized secreted protein, than cells stimulated at 0 h (P less than 0.001). The acquisition of increased responsiveness was not abrogated by washing and resuspending the cells in fresh medium. Since the increased responsiveness could be inhibited by the addition to the cultures of small numbers of cells previously activated by Con A it is suggested that the enhanced reactivity acquired in culture represents the loss of a subpopulation of suppressor cells that modulate the T-lymphocyte response. Cells from nine patients with active, untreated systemic lupus erythematosus demonstrated normal responses to optimal concentrations of Con A added at 0 h, but an impaired response to Con A, 1 mug/ml. When these cells were incubated for 24 h, a significant increased response to Con A was not observed. This observation suggests that patients with active SLE lack circulating suppressor cells. When seven SLE patients were again studied after corticosteroid therapy had led to clinical improvement, the response to Con A, 1 mug/ml, added after 24-h incubation was similar to that observed in normal controls, suggesting that suppressor function in SLE returns as disease activity declines.

Human mononuclear cell factors mediate cartilage matrix degradation through chondrocyte activation.

Human blood mononuclear cells (BMC) in short-term culture secrete one or more factors that induce degradation of matrix proteoglycan and collagen in cartilage explants in organ culture. Induction of matrix degradation took place both in nasal septum and articular cartilage explants in the presence of the mononuclear cell supernates. Cartilage degradation in this system was absolutely dependent on the presence of live chondrocytes. Matrix depletion did not occur in dead cartilage explants cultured with active supernates. Supernates obtained from unstimulated BMC showed variable cartilage matrix degrading activity (MDA). BMC stimulated with phytohemagglutinin (PHA) showed increased MDA, which in one dilution experiment was found to be five times higher than that in the unstimulated control supernate. Concanavalin A and pokeweed mitogen were also shown to stimulate release of MDA. Time experiments showed that most of the degrading activity was released by the mononuclear cells during the first day of culture. The cellular origin of MDA was investigated with the aid of partially purified BMC subpopulations. Removal of adherent cells resulted in a decrease of MDA release. Purified T lymphocytes failed to show enhanced MDA release in spite of their ability to mount a virtually intact proliferative response to PHA. Purified adherent cells also failed to show enhanced PHA-dependent MDA release. Nevertheless, restoration of PHA-dependent MDA release took place in reconstituted cell populations containing both T lymphocytes and monocytes. These experiments suggest that MDA may be released by adherent mononuclear cells, presumably monocytes, and that the PHA-dependent increase in MDA release may be mediated by T lymphocytes. Partial characterization of MDA by gel chromatography showed one active fraction corresponding to an apparent molecular weight ranging from 12,000 to 20,000. The fraction was also shown to degrade cartilage matrix only in the presence of live chondrocytes. These results demonstrate that factors released by human BMC mediate degradation of matrix proteoglycan and collagen in intact cartilage explants through chondrocyte activation. This pathogenic mechanism may play a role in in vivo cartilage destruction in chronic inflammatory joint diseases.

Mechanisms of disruption of the articular cartilage surface in inflammation. Neutrophil elastase increases availability of collagen type II epitopes for binding with antibody on the surface of articular cartilage.

We recently observed that specific antibodies to type II collagen do not bind in appreciable amounts to the intact surface of articular cartilage, whereas antibodies to the minor collagen types V, VI, and IX do. These results suggest that the outermost cartilage surface layer prevented interaction of the antibodies with the major collagen type in articular cartilage. The present studies were designed to investigate the pathogenic mechanisms involved in the disruption of the cartilage surface layer in inflammatory arthritis. Articular cartilage obtained from rabbits undergoing acute antigen-induced arthritis of 72 h duration showed a significant increase in binding of anti-type II antibody to cartilage surfaces compared with normal control cartilage (P less than 0.01). Augmentation of anti-type II binding was also observed upon in vitro incubation of bovine articular slices or intact rabbit patellar cartilage for 1 h with human polymorphonuclear neutrophils (PMN), PMN lysates, or purified human PMN elastase. This increase was not inhibited by sodium azide, nor was it enhanced by incubation of cartilage with the strong oxidant hypochlorous acid. Chondrocyte-mediated matrix proteoglycan degradation in cartilage explants cultured in the presence of cytokines failed to increase antibody binding appreciably. The augmentation in antibody binding seen with PMN lysates was inhibited by the nonspecific serine-esterase inhibitor PMSF, but not by the divalent metal chelator EDTA. The elastase-specific inhibitor AAPVCMK also inhibited most of the PMN-induced increase in antibody binding, whereas the cathepsin G-specific inhibitor GLPCMK was much less effective. Incubation of intact cartilage with purified human PMN elastase indicated that this serine esterase could account for the increase in anti-type II collagen antibody binding to intact cartilage surfaces. These studies suggest that in an inflammatory response, PMN-derived elastase degrades the outer layer of articular cartilage, exposing epitopes on type II collagen. They also help clarify the pathogenic mechanisms involved in early articular cartilage damage in inflammatory joint diseases.

Rheumatoid hyperviscosity syndrome

Two patients with rheumatoid arthritis and a serum hyperviscosity syndrome are described. Both patients had nodular rheumatoid arthritis, weakness and dyspnea, bleeding diathesis, a striking purplish red palmar erythema and high serum viscosity. Their serum had high titers of rheumatoid factor and large amounts of intermediate IgG complexes. Viscosity measurements of the isolated protein fractions suggested that the high serum viscosity was due to the interaction of rheumatoid factor with the intermediate IgG complexes, resulting in the formation of large molecular conglomerates. Viscosity measurements following recombination of the isolated protein fractions, the effects of sulfhydryl reagents on the viscosities measured and the determination of intrinsic viscosities confirmed this hypothesis.

In vitro synthesis of immunoglobulins and IgM-rheumatoid factor by blood mononuclear cells of patients with rheumatoid arthritis.

SummaryIn vitro synthesis of IgG, IgM and IgM-rheumatoid factor (IgM-RF) was investigated in unstimulated and pokeweed mitogen (PWM)-stimulated 7-day cultures of blood mononuclear cells (BMC) from 28 seropositive patients with active rheumatoid arthritis (RA), in a second group of 13-day cultures from 94 unselected rheumatoid patients, and in 21 healthy controls. Both normal and rheumatoid BMC cultures synthesized IgM-RF in response to PWM stimulation. Mitogen-induced stimulation was shown to be dependent on the presence of T-lymphocytes. PWM-induced IgM and IgM-RF synthesis were reduced in BMC from rheumatoid patients in comparison with healthy controls. However, the fraction of IgM-RF in the total IgM synthesized was significantly higher in the RA supernatants than in the controls, suggesting the presence of a larger precursor B-cell population committed to IgM-RF synthesis in those cultures.BMC from 44 of the 94 patients demonstrated spontaneous synthesis of IgM-RF, and this was positively correlated with disease activity and rheumatoid factor titer. Spontaneous production was shown to reside in the T-lymphocyte depleted, adherent cell-depleted, B-cell subpopulation.It is concluded that in active RA there is a specific activation and expansion of the circulating B-cell subpopulation committed to IgM-RF synthesis, possibly due to abnormal immunoregulatory mechanisms modulating synthesis of this antibody.

Relationship Between Soluble Collagen and Urinary Hydroxyproline in the Lathyritic Rat.

Summary Urinary excretion of hydroxyproline of weanling rats, rendered lathyritic by daily injection of beta-aminopropionitrile, was 3 times greater than that of controls. A parallel increase in the neutral salt extractable collagen of the skin was observed. No significant change was found in the total collagen of the skin. It is concluded that the increase in urinary hydroxyproline observed is related to the increased size of the metabolically active, soluble collagen pool.

OXIDATIVE MODIFICATION OF INFLAMMATORY SYNOVIAL FLUID IMMUNOGLOBULIN G

The release of highly reactive oxygen-derived products by activated phagocytic cells in inflammatory foci plays a major role in defense mechanisms against infection and in the generation of tissue injury. Oxidative modification of proteins in inflammatory foci may give rise to products that contribute to the perpetuation of inflammation. The present studies analyze the oxidative alterations of inflammatory synovial fluid immunoglobulin G (IgG). IgG was purified from the synovial fluids of five patients with rheumatoid arthritis and two patients with acute gouty arthritis by three sequential steps: gel filtration chromatography, immunoaffinity chromatography, and a final gel filtration chromatography step under dissociative conditions (4 M guanidine). The resulting protein peaks of > 150 kDa (pool I-1), 150 kDa (pools I-2 and II-2), and < 150 kDa (pools I-3 and II-3) were tested for the presence of a fluorescence profile distinctive of oxidized proteins, the peak corresponding to monomer IgG (pool II-2) was subjected to quantitative amino acid analysis, and the results were compared with a standard preparation of normal IgG oxidized with HOCl. In addition, the protein pools were tested for the presence of lipid peroxide products. The results indicate that a portion of the affinity-purified IgG formed high-molecular-mass covalently cross-linked aggregates as evidenced by its presence in the >150-kDa pool after dissociative gel filtration chromatography. Moreover, this fraction and the pool corresponding to monomer IgG (pool II-2) exhibited the fluorescence profile characteristic of oxidized proteins. Amino acid analysis of the monomer IgG fraction revealed decreases in the content of histidine, methionine, tyrosine, and cystsine, which were similar to the alterations measured in normal IgG oxidized by HOCl. The synovial fluid and standard oxidized IgG showed the presence of oxidative by-products of tyrosine (monochlorotyrosine) and cysteine (cysteic acid). The synovial fluid IgG yielded a novel component that was not present in the standard control or oxidized IgG. This component was partially identified by mass spectrometry. Finally, the smaller peptide fraction isolated from affinity-purified synovial fluid IgG (pools I-3 and II-3) only after the gel filtration chromatography step under dissociative conditions exhibited evidence of oxidative damage and the presence of high concentrations of thiobarbituric acid-reactive material (TBAR). These observations suggest that oxidative processes in inflammatory foci generate products derived from protein and lipids that may contribute to the self-perpetuation of inflammation.

Immune mechanisms in osteoarthritis

0 STEOARTHRITIS (OA) is generally thought of as a disease of articular cartilage failure induced by a variety of genetic, metabolic, and biomechanical factors. Coexisting with the cartilage abnormalities, most affected joints exhibit synovial inflammatory responses that may range from minimal lining cell layer hyperplasia to severe acute and chronic synovitis. The synovial response may be responsible for the pain symptomatology associated with early OA. Although it is unlikely that immune processes play an important primary role in initiating synovial inflammation, cellular and humoral immune responses to cartilage macromolecules may constitute important factors in the maintenance and severity of synovitis in OA. This review focuses on the specific immunologic mechanisms that may be operative in OA. The physiopathogenesis of inflammation induced by immunologic mechanisms is not discussed in detail because it is likely that once the antigenspecific interactions occur, the development of the inflammatory process results from the generation of cellular and humoral proinflammatory factors that constitute a final common pathway to most inflammatory processes, regardless of the disease entity under consideration.

Synthesis of rheumatoid factor in vitro: implications for the pathogenesis of rheumatoid arthritis.

R HEUMATOID FACTOR (RF), an autoantibody that has the ability to bind to the Fc portion of immunoglobulin is present in the serum of the majority of adult patients with rheumatoid arthritis (RA). This autoantibody is clearly associated with RA, yet its role in the disease process is not well understood.‘m3 The most severe and erosive forms of RA are usually associated with high titers of RF.4 However, the presence of RF has been reported in normal individuals and in diseases other than RA.’ This evidence suggests that RF is only one of multiple interacting factors in the etiology of RA, perhaps requiring a susceptible host for full disease expression. In most clinical settings, RF levels in serum are measured by using agglutination methods with antibody coated latex particles or sheep RBCS.~ Recently, sensitive immunoassays for RF have been developed that permit measurement of RF production by cell cultures in vitro. The earliest of these was a direct hemolytic plaque assay specific for RF,’ which allowed for enumeration of RF producing cells. This was followed by solid-phase immunoassays using radiolabeled or enzyme linked markers*-” to measure RF secretion by cells in tissue culture. When used to measure serum levels of RF, these assays generally correlate with routine laboratory methods such as the latex agglutination test. However, they are sensitive to nanogram quantities of secreted antibody. Thus, it has become

The relationship between circulating CD5+ B lymphocytes and in vitro autoantibody synthesis in normal individuals

Recent observations suggest that a subpopulation of B lymphocytes bearing the phenotype CD5+ may be enriched for cells committed to the synthesis and secretion of autoantibodies. We had previously shown that a subset of normal individuals has an expanded subpopulation of B lymphocytes committed to the synthesis of IgM and IgM-rheumatoid factor (RF), and that this condition was associated with HLA-DR4 (4). In these studies, we performed simultaneous quantitation of the size of the circulating CD5+ B lymphocyte subpopulation in a group of 20 normal donors, and of the pokeweed mitogen-induced in vitro synthesis of a panel of autoantibodies by the same peripheral blood cells depleted of CD8+ suppressor lymphocytes in 18 of the 20 individuals. The culture supernatants were assayed for total IgM and IgG, RF, IgM, and IgG anti-single-stranded DNA, anti-human thyroglobulin, and anti-tetanus toxoid. The mean percentage CD5+ B cells was 13.5 +/- 2.5%. There was no significant correlation between total B lymphocytes and CD5+ B cells (R = 0.25, P greater than 0.20. Positive correlations were found between the proportion of circulating CD5+ B lymphocytes and synthesis of RF (R = 0.73, P less than 0.001), and IgM anti-DNA (R = 0.58, P less than 0.03). Significant correlations were not found between CD5+ B cells and secreted IgM or IgG antibodies against the exogenous antigen tetanus toxoid, measured in the same supernatants. The antibodies produced in vitro by T cell-dependent B cell activation appear to have limited or no polyspecificity. These results indicate that the size of the circulating CD5+ B cell subpopulation in any given individual contributes importantly to the magnitude of autoantibody synthesis in cultures where T cell-mediated B lymphocyte activation takes place in the absence of suppressor signals.