IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular sources.

We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays. IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line. IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting. The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera. In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay. Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay. Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml). No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6. The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6. Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied. Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation. Similarly, there was no constitutive production of IL-1 by these cells. However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS. The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells. These findings suggest that IL-6 is involved in inflammatory joint disease. However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate.

Sickled cells in synovial fluid: clue to unsuspected hemoglobinopathy.

In this report, we have described three patients who had hemarthroses, with sickled red blood cells discovered by analysis of synovial fluid. On the basis of this observation, each patient was evaluated for the presence of abnormal hemoglobins, and each was found to have a hemoglobinopathy that was previously unsuspected. These patients differ from those in other reports in that two of the three had no associated arthritic condition that could readily explain synovitis or a condition that predisposed them to bleeding into a joint. Although the accumulated evidence suggests that heterozygous hemoglobinopathies do not produce arthritic syndromes, these reports again raise that question. We cannot conclude, however, that the hemarthroses were definitively caused by the underlying hematologic abnormality. Important when synovial fluid is mixed with blood, since other medical conditions can be diagnosed if abnormal findings are detected.

Acute monoarthritis associated with intracellular positively birefringent Maltese cross appearing spherules.

A 57-year-old black woman presented with acute monoarthritis of the left knee. Numerous intra and extracellular strongly positively birefringent lipid spherules with a Maltese cross appearance were seen in the synovial fluid from the affected joint when viewed under compensated polarized light microscopy. This represents the 7th reported case of atraumatic acute arthritis associated with large numbers of positively birefringent lipid spherules. We review previous case reports and discuss the present understanding of the pathogenesis and the significance of the association of large numbers of birefringent lipid spherules with acute arthritis.

Ischemia of the femoral head in Perthes' disease: is the cause intra- or extravascular?

We examined selected parameters of the clotting and fibrinolytic system of 26 boys with aseptic necrosis of the femoral head and then evaluated the pressure of the fluid in the cavity of the hip joint with the help of ultrasonic examination. No disturbances were discovered in the coagulation system and ultrasonography ruled out the possibility that extravascular pressure had caused the necrosis. We confirmed a significantly greater level of alpha 1-antitrypsin in comparison with the control group, which may indicate a decrease in fibrinolytic activity and confirm the hypothesis that there is an intravascular pre-disposition towards the appearance of clots in the vascular system of the femoral head in patients with Perthes' disease.

Autoreactive T cells in rheumatic disease (1). Analysis of growth frequencies and autoreactivity of T cells in patients with rheumatoid arthritis and Lyme disease.

A limiting dilution system was established in order to estimate frequencies of interleukin-2 (IL-2)-responsive, autoreactive and alloreactive T cells in samples of peripheral blood (PBL) and synovial fluid lymphocytes (SFL), from patients with rheumatoid arthritis (RA) and lyme disease, as well as from healthy donors and a patient with osteoarthrosis. The frequencies of IL-2-dependent T-cell colony formation were significantly higher in patients with RA and lyme disease (median: 1/287) as compared to controls (median: 1/1,313) indicating a preactivation of T cells in these patients in vivo. Autoreactivity was measured by the proliferative response of T-cell lines to autologous irradiated PBL as stimulating cells. The frequencies of autoreactive T cells in blood were significantly higher in patients (median: 1/2,615) as compared to controls (median: 1/19,607). There was no significant difference in autoreactive T-cell frequencies between the patients' SFL (median: 1/3,185) and PBL (median: 1/2,615). In every case the frequency of alloreactive T cells exceeded the frequency of autoreactive T cells. Most autoreactive T-cell lines were also alloreactive and were shown to be MHC Class II-restricted. There is evidence of a down regulation of autoreactive T cells by suppressor cells in peripheral blood in two cases with elevated autoreactive T-cell frequencies (one RA patient and one control patient suffering from a viral infection). In contrast, no suppression of autoreactive T cells was observed in the RA patients' SFL or in PBL and SFL from patients with lyme disease. These results suggest that the chronic inflammation observed in RA and lyme disease may be supported by an elevated number of autoreactive T cells in the absence of suppressive mechanisms.

The preferential accumulation of helper-inducer T lymphocytes in inflammatory lesions: evidence for regulation by selective endothelial and homotypic adhesion.

The mechanisms which lead to the accumulation of T lymphocytes into inflammatory lesions are not clearly understood. We have previously shown that synovial CD4 T lymphocytes are mostly CDw29+UCHL1+ (helper-inducer cells) and very few carry the CD45R antigen which identifies the suppressor-inducer subset. Synovial CD8+ cells are also CDw29+UCHL1+CD45R-. In the present study, lymphocytes from pleural and peritoneal inflammatory infiltrates were shown to have a similar phenotypic pattern. Furthermore, it was demonstrated that the CDw29+UCHL1+ subset had a greater ability than CD45R+ cells to adhere to endothelial cells and to form homotypic clusters. Differential surface expression of LFA-1 on the two subsets was also shown, but this could not account for the demonstrated adhesion differences. Differences in adhesion between CDw29+/UCHL1+ and CD45R+ cells may explain the preferential accumulation of CDw29+/UCHL1+ cells in inflammatory infiltrates and underlie some of the functional differences between cells taken from sites of chronic inflammation and those from peripheral blood.

Interleukin-1 production by mononuclear cells from rheumatoid synovial effusions.

The polypeptide interleukin-1 (IL-1) is a cytokine that may mediate inflammation and connective tissue damage in rheumatoid arthritis (RA). We examined cytokine production by normal blood and by rheumatoid synovial mononuclear cells with sensitive (picomolar) assays. The assays were immunolabeling and immunoblotting with rabbit anti-IL-1 beta sera, and proliferation of the murine D10 cell line to IL-1. Little or no cytokine was detected in rheumatoid joint fluid or in exudate mononuclear cells from patients with acute rheumatoid flares. The mononuclear cells could be induced to make IL-1 upon stimulation with lipopolysaccharide (LPS). The responsive cells were monocytes, since all could be double-labeled with anti-IL-1 and the monocyte-specific CD14 antibody. More than 80% of the synovial fluid monocytes made IL-1 beta after 24 hr in 2 ng/ml LPS. Other agents failed to induce IL-1 from enriched populations of monocytes including interferon gamma (IFN-gamma), poly (I/C), phorbol myristate acetate (PMA), concanavalin A (Con A), phytohemagglutinin (PHA), and anti-CD3 antibodies. Relatively high levels of dendritic cells (DC) were present in RA effusions, but these did not produce IL-1 in response to any of the above stimuli. Blood dendritic cells also did not make IL-1, whereas blood monocytes responded comparably to synovial exudate cells. The data indicate that rheumatoid exudate monocytes make very little IL-1 during acute flares of arthritis and that this cytokine is primarily a macrophage rather than a dendritic cell product.

Increased macrophage division in the synovial fluid of goats infected with caprine arthritis-encephalitis virus.

Macrophages are a major component of the arthritic lesions induced by the lentivirus caprine arthritis-encephalitis virus (CAEV). Using autoradiography and the appearance of mitotic figures to detect dividing macrophages, we found that 2.1% +/- 0.2% of synovial fluid macrophages from uninfected goats are dividing and that after infection with CAEV the percentage increases three- to sixfold. The enhanced macrophage division was not associated with increased dividing of blood monoblasts. The amount of macrophage division correlated with two measures of arthritis: joint swelling and the number of synovial fluid macrophages. Induction of an immune response in the joints of CAEV-infected goats increased the number of dividing macrophages. The synovial fluid of infected animals was mitogenic for macrophages from infected animals in amounts that correlated with the amount of macrophage division occurring in the joints. Activated lymphocytes produced nondialyzable lymphokines mitogenic for macrophages from CAEV-infected goats but not from uninfected goats. These results suggest that in situ macrophage division contributes to the lesions induced by CAEV and that infection leads to greater responsiveness of macrophages to mitogenic factors produced by lymphocytes.

Geriatric knee disorders, Part I: Evaluative techniques.

It is important to remember that knee disorders seen in the elderly are distinctly different from those seen in the younger individual. In the elderly, the problems are generally the result of chronic processes and, occasionally, an acute process on top of a chronic disorder. A careful history and physical examination should enable the physician in most cases to make the correct diagnosis. However, at times, specific laboratory studies may be ordered to confirm the diagnosis. In part I of this two-part review of geriatric knee disorders, the authors will focus on the anatomy, history, and physical examination of the knee joint. Common knee disorders specific to the elderly will be discussed in part II.

Intraarticular methylprednisolone therapy in hemophilic arthropathy.

This small pilot study examined the use of intraarticular methylprednisolone in hemophilic synovitis. Nineteen joints in ten adult hemophiliacs were studied. There was subjective improvement at 24 hr following injection in 79% of joints injected, and the improvement persisted up to 8 wk in 58%. The number of hemarthroses decreased following intraarticular steroids (mean of 7.7 bleeds in the 8 wk prior to injection versus a mean of 1.9 bleeds in the 8 wk following injection). Similarly the amount of clotting factor used for the injected target joint decreased from a mean of 7,616 units to 2,315 units postinjection (p less than .001). Improvement correlated with presence of synovitis but not with radiologic stage of the joint. Aspirated synovial fluids were analyzed and showed characteristics consistent with low-grade inflammation. These preliminary observations suggest that intraarticular corticosteroid injection may be a useful therapeutic tool in the medical management of hemophilic arthropathy.

Cholesterol crystals in shoulder synovial fluid.

Samples of 1945 synovial fluids have been examined from patients with a wide variety of joint disorders. Typical cholesterol crystals were seen in only 14 of these samples, all from the shoulders of six patients with severe rheumatoid arthritis and persistent shoulder effusions.

Functional and phenotypical characterization of activated T cells from intra-articular sites in inflammatory joint diseases. Possible modulation of the CD3 antigen.

The presence of activated T lymphocytes bearing interleukin 2 (IL-2) receptors and HLA class II (Ia) antigens accompanied by impaired T cell functions such as a decreased mitogenic responsiveness are characteristic findings, especially in intra-articular sites in chronic inflammatory joint diseases. The objective of the present study was to further characterize these in vivo activated T cells by the investigation of IL-2 production and a possible T cell receptor modulation. IL-2 receptors were found to be expressed primarily in the CD4+ subset. The Ia+ subset expressing both DR and DQ antigens showed a weaker mitogen-induced response as compared to the Ia- fraction. A decreased mitogen-induced IL-2 production and a lower response to anti-CD3 monoclonal antibodies was observed with synovial T lymphocytes as compared to peripheral blood T cells. The density of the CD3 molecule, known to be closely associated with the T cell receptor, was significantly lower in intra-articular sites, while other T cell-specific surface molecules were expressed to a similar extent in both compartments. The decreased synovial T cell mitogenesis was not restored by the addition of lymphokines (IL-1 and IL-2) or blood monocytes, nor by removing CD8+ T cells. These data present further evidence for a significant T cell activation in intra-articular sites in chronic inflammatory joint diseases. The decreased expression of the CD3 glycoprotein suggests a modulation by so far unidentified antigen(s), which could also be responsible for the weak T cell response elicited by polyclonal mitogens.

Abnormal distribution of the helper-inducer and suppressor-inducer T-lymphocyte subsets in the rheumatoid joint.

T lymphocytes can be divided into two main phenotypic populations, CD4 and CD8. These can be further subdivided into 2H4, 4B4, or UCHL1 subsets by appropriate monoclonal antibodies. We have investigated these subsets in the synovial fluid of patients with rheumatoid arthritis and have found (i) a virtual absence of CD4+ 2H4+ and the marked reduction of CD8+ 2H4+ T cells; (ii) a marked increase of CD4+ 4B4+ and CD8+ 4B4+ T cells; and (iii) a marked increase of CD4+ UCHL1+ and CD8+ UCHL1+ T cells compared with peripheral blood. Although the functions of the CD8 subsets are not known, the virtual absence of CD4+ 2H4+ suppressor-inducer T cells and the marked increase of CD4+ 4B4+ helper-inducer T cells and of CD4+ UCHL1+ memory T cells may help to explain the many known functional immunological properties of synovial T cells.

A urine inhibitor of interleukin 1 activity affects both interleukin 1 alpha and 1 beta but not tumor necrosis factor alpha.

Urine from monocytic leukemia and other febrile patients contains an inhibitor of interleukin 1 (IL-1), as measured by prostaglandin E2 and collagenase production by human fibroblasts and synovial cells. With the use of recombinant IL-1, the IL-1 inhibitor was partially purified by using ammonium sulfate precipitation, anion-exchange, and gel filtration chromatographies. IL-1 inhibitory activity elutes with an 18,000 to 25,000 apparent molecular size. The same fractions also inhibit IL-1 assayed by the proliferation of murine thymocytes and human fibroblasts. Both forms of human recombinant IL-1, IL-1 alpha and IL-1 beta, which show only 26% homology, but nevertheless bind to the same receptor, are affected by this natural inhibitor to the same extent. In contrast, human recombinant tumor necrosis factor, which shares some of the biologic activities of IL-1, is not inhibited by the urinary IL-1 inhibitor. This study shows that the various biologic activities of both forms of human recombinant IL-1 are inhibited by a partially purified natural urine-derived factor.

Mononuclear cells in synovial fluid of rheumatoid arthritis patients undergoing joint surgery.

Analysis of synovial fluid from patients with rheumatoid arthritis (RA) and a long history of RA, who were undergoing synovectomy and joint surgery, revealed that 71 +/- 15% of the mononuclear cells in the synovial fluid were lymphocytes. 3 +/- 2% lymphoid blast cells, and 26 +/- 15% of monocytes/macrophages. The lymphocyte population consisted mainly of T cells, and 72 +/- 15% of lymphocytes were OKT11-positive. OKT8-positive cells (48 +/- 9%) dominated over OKT4-positive (32 +/- 8%) cells with a mean OKT4/OKT8 ratio of 0.69. On the basis of Ia- and Tac-markers, very few of the lymphocytes were activated. Few B cells (12 +/- 7%) and no plasma cells were seen. The results are discussed in relation to previous reports.

Inhibition of prostaglandin biosynthesis by etodolac. I. Selective activities in arthritis.

Etodolac is the first anti-inflammatory drug belonging to the tetrahydropyranoindole class. In contrast to several other common anti-inflammatory drugs, etodolac exhibited an unusually high potency as an inhibitor of established adjuvant arthritis relative to its activity against carrageenan paw edema in the rat. This phenomenon led us to investigate whether the ability of NSAIDs to inhibit prostaglandin biosynthesis differed between cultures of macrophages, which are present in inflammatory exudates, and cultures of synoviocytes and chondrocytes, which contribute to inflammation of the articulating joint. Although other anti-inflammatory drugs were found to be equally active in all three cell types, etodolac was found to be much more effective on the cells of the joint than on the macrophage. This differential activity may be responsible for the striking efficacy of etodolac as an anti-arthritic drug.