Lack of immune response to methyl methacrylate in lymphocyte cultures.

To analyze whether or not methyl methacrylate is immunologically inert, peripheral blood mononuclear cells were cultured with finely pulverized methyl methacrylate. Phytohemagglutinin (PHA) lectin, purified protein derivate of tuberculin (PPD) antigen, and culture medium alone were used as positive and negative controls. Lymphocyte kinetics on culture Days 0, 1, 3, and 5 were studied. Major histocompatibility complex locus II antigen (MHC locus II antigen; Ia) and interleukin-2 receptor (IL-2R; Tac) expression were analyzed using the avidin-biotin-peroxidase complex (ABC) method and lymphocyte DNA synthesis using 3H-thymidine incorporation and beta-scintillation counting. On culture Days 1 and 3, lymphocytes and monocytes were seen under the light microscope to be attached to methyl methacrylate particles. However, the results disclosed no methyl methacrylate-induced DNA synthesis, although methyl methacrylate-induced MHC locus II antigen and IL-2R activation marker expression were recorded; notably, this expression was less pronounced than that seen in PHA or PPD antigen driven lymphocyte response. The results suggest that methyl methacrylate is essentially an immunologically inert implant material. However, it seems to induce inflammatory mononuclear cell migration and adhesions leading to slightly nonspecific lymphocyte reaction.

Primary inflammatory reaction in synovial fluid and tissue in rabbit immobilization osteoarthritis.

The kinetics and composition of the primary cellular inflammatory process were studied in the synovial fluid (SF) and synovial tissue (ST) compartments of a rabbit knee immobilization osteoarthritis model. Immobilization induced rapid migration of neutrophils (59% +/- 26% of all cells) into SF in three days, which was accompanied by nonspecific esterase-positive monocytes (71% +/- 8% of all mononuclear cells). This finding suggests that non-specific inflammation mediated by phagocytic leukocytes predominates the cellular response in the SF compartment. In contrast, morphometric analysis of ST proper showed an inflammatory mononuclear cell response, the intensity of which diminished over time during the study period from Day 3 (416 +/- 59 cells per 0.049 mm2 ST tissue) through Day 10 (305 +/- 32 cells) to Day 35 (174 +/- 36 cells). A dotlike T-pattern alpha-naphthyl acetate esterase (ANAE) was found in the T-cell-dependent areas of secondary lymphatic tissue in the spleen, enabling immunocytologic ANAE marker studies. The ST response in situ was predominated by tissue macrophage, though infiltrates rich in T lymphocytes were present in the immediate sublining stroma. There was a significant correlation between the intensity of the SF cell response (total recovery) and the percentage of neutrophils, but there was no correlation between the intensity of the ST response and the proportion of T lymphocytes. These T-cell accumulations together with the local proliferation of fibroblastlike lining cells and stromal fibroblasts suggest that the primary inflammatory cell response is not caused by either wear and tear or mechanically by cartilage fragments.

Chemoattractant production by synovial fluid cells in chronic arthritis. A study with a new double-chamber method for quantitating the motility of mononuclear cells labelled with chromium-51.

A new method was developed to study the migration of lymphocytes under in vitro conditions. Attractant fluid was added to the appropriate lower wells, which were filled to the brim. Peripheral blood mononuclear cells were labelled with 51Cr and then put into the upper wells. Surface tension and capillary force are enough to make clamping of these plates possible, separated by a polycarbonate filter, 15 microns thick with a pore size of 8 microns, between these two standard multiwell microculture plates. After three hours of incubation at +37 degrees C in a humidified 5% CO2 atmosphere, the migrated cells were harvested from the lower wells using a semiautomatic harvester, and the radioactivity was counted. The present method makes it unnecessary to enrich or select for different cell subsets for migration studies. Instead, density gradient separated mononuclear cells as such can be used to assess their migratory capacity.

Aggressive granulomatous lesions associated with hip arthroplasty. Immunopathological studies.

The local immunopathological response was analyzed in six patients who had a revision of a total hip prosthesis because of an aggressive granulomatous lesion and in six patients who had a revision because of common loosening of the prosthetic stem. All twelve patients had had a total replacement arthroplasty for primary osteoarthrosis. All of the prostheses had been cemented. The aggressive granulomas consisted of well organized connective tissue containing histiocytic-monocytic and fibroblastic reactive zones. The granulomas were highly vascularized, and villous structures were observed at many sites. In contrast, the areas around the loose cemented stems were characterized by dense connective tissue. Immunohistological evaluation revealed that most of the cells in the aggressive granulomatous tissue were multinucleated giant cells and C3bi-receptor and nonspecific esterase-positive monocyte-macrophages. This cytological finding suggests a foreign-body-type reaction, compatible with the rapidly progressive lytic nature of the lesion that was shown radiographically. There was a clear-cut difference between aggressive granulomatosis and the more common lesion accompanying prosthetic loosening--namely, the relative lack of activated fibroblasts in granulomatosis. We suggest that granulomatosis involves an uncoupling of the normal sequence of monocyte-macrophage-mediated clearance of foreign material and tissue debris that is normally followed by fibroblast-mediated synthesis and remodeling of the extracellular matrix. We also suggest that aggressive granulomatosis in association with a cemented hip prosthesis is a distinct entity, not only clinically and radiographically, but also histopathologically.

Herpes simplex virus antigens and inflammatory cells in oral lesions in recurrent erythema multiforme. Immunoperoxidase and autoradiographic studies.

Herpes simplex virus (HSV) antigens were sought in 15 biopsy specimens from both lesional mucosa and clinically healthy looking oral mucosa between attacks in patients with erythema multiforme (EM). Four of the eight biopsy specimens obtained from lesional EM mucosa stained positively with HSV-1-and/or HSV-2-specific antisera applied in direct immunoperoxidase staining. Of the 16 tissue specimens used as controls, two displayed positive staining with HSV-1 and/or HSV-2. Five of the seven biopsy specimens from macroscopically healthy oral mucosa obtained between attacks from patients with recurrent EM stained positively with HSV-1 and/or HSV-2. Of the six tissue specimens used as controls, three stained positively. Most of the local inflammatory mononuclear cells belonged to the T cell series, mainly to the CD-4 subset. A small proportion of the local T cells were blast transformed as assessed by CD-25 expression and [3H]thymidine incorporation. This, together with the findings showing a lower degree of activation in the biopsy from macroscopically healthy looking mucosa between attacks suggest an active role of the cell-mediated immune response in the genesis of oral lesions in EM. The persistence of HSV antigens, and the well-established role of HSV as a precipitating factor in recurrent EM, suggest that HSV may be involved, but since HSV seems to be present in other mucosal lesions as well as in clinically healthy mucosa, quite frequently an additional, hitherto unknown factor must be present in order that EM may occur.

Localisation of lysozyme mRNA in rheumatoid synovial membrane by in situ hybridisation.

Type A synovial lining cells have been shown to contain lysozyme in their lysosomes. This might be phagocytosed because synovial fluid contains lysozyme originating from tissue macrophages and articular cartilage but in arthritides, in particular, from neutrophils. In situ hybridisation with 35S labelled cDNA was used to detect mRNA for lysozyme over synovial lining in patients with rheumatoid arthritis. No hybridisation was found with lactoferrin cDNA, which was used as a negative control. Computer search against the EMBL gene bank (release 14) did not show any significant cross hybridisation to a known sequence. In cytological specimens 35S-cDNA:mRNA hybrids were observed in positive but not in negative control cells. The presence of lysozyme and its mRNA suggests that type A synovial lining cells are of mononuclear phagocyte lineage.

Differences in responses of normal and rheumatoid arthritis peripheral blood T cells to synovial fluid and peripheral blood dendritic cells in allogeneic mixed leukocyte reactions.

We examined the response of normal T cells to dendritic cells isolated from the synovial fluid (SF) of patients with either rheumatoid arthritis (RA) or seronegative spondylarthropathies (rheumatoid variants) and to dendritic cells from normal and RA peripheral blood (PB) in the allogeneic mixed leukocyte reaction. Despite the differences in the response kinetics, the stimulatory capacity of SF dendritic cells was similar to that of PB dendritic cells in a 7-day mixed leukocyte reaction. We also tested the responsiveness of normal and RA PB T cells to various allogeneic dendritic cells and found that RA PB T cells responded poorly to both rheumatoid variant SF dendritic cells and normal PB dendritic cells. However, when dendritic cells from RA SF were used as stimulators, the response of RA PB T cells was significantly greater than that of normal PB T cells (P less than 0.02). This difference in response was explained in part by a proliferation of the CD8 T cell subset. There was also a shift of low-intensity CD4+, CDw29+ cells to high-intensity CD4+, CDw29+ cells seen in RA PB T cells but not in normal PB T cells, by fluorescence-activated cell sorter analysis.

Cytokines in chronic inflammatory arthritis. III. Rheumatoid arthritis monocytes are not unusually sensitive to gamma-interferon, but have defective gamma-interferon-mediated HLA-DQ and HLA-DR induction.

Macrophages present in the synovium and synovial fluid of patients with rheumatoid arthritis (RA) express large amounts of HLA-DR molecules on their surface, despite low levels of gamma-interferon (gamma-IFN) in the joint. To determine whether this apparent paradox is the result of increased sensitivity to gamma-IFN in RA, we compared concentrations of gamma-IFN that induced HLA-DR and DQ on peripheral blood monocytes of RA patients and normal donors, using fluorescence-activated cell sorter analysis. Among normal donors, highly variable sensitivity to gamma-IFN was observed. Higher amounts of gamma-IFN were required to induce class II major histocompatibility complex molecules on RA monocytes versus normal monocytes. The maximum amount of HLA-DR that could be induced on RA and normal monocytes was similar; however, peak levels of HLA-DQ were significantly less in RA. Monocytes from patients with other forms of chronic inflammatory arthritis had intermediate HLA-DQ expression after gamma-IFN treatment. These data suggest that an increased sensitivity to gamma-IFN in RA does not account for the high level of HLA-DR expression in the joint. Also, a defect in HLA-DQ and HLA-DR induction by gamma-IFN was observed.

Dendritic cells in health and disease.

Cells with a dendritic morphology that are rich in surface class II histocompatibility antigens (Ia antigens) but lacking in other lymphocytes or monocyte markers constitute a small (less than 1%) proportion of circulating mononuclear cells, but in inflammatory joint effusions they comprise up to 7% of the mononuclear population. Their role as accessory cells in normal autologous and allogenic mixed leukocyte reactions is reviewed and the possible contribution of dendritic cells to intra-articular immunologic processes is considered.

Presence of platelet glycoproteins Ib and IIb-IIIa in inflammatory and noninflammatory synovium.

Rheumatoid synovial tissue and noninflammatory synovial tissue from patients with meniscus lesions were stained using monoclonal antibodies against platelet 150 kDa Ib glycoprotein (gp Ib) and against 140/110 kDa IIb-IIIa glycoprotein complex (gp IIb-IIIa) applied with the avidin-biotin-peroxidase complex method. Gp Ib and gp IIb-IIIa positive intravascular platelet aggregates were not seen, except locally in the capillary blood vessels of one rheumatoid synovial sample. This suggests that the platelets and the clotting sequence are not activated in inflamed synovial tissue. However, in many of the synovial capillaries endothelial immunoreactivity was seen. This reaction could have been due to cross reaction, since the vitronectin receptor beta chain is structurally identical to platelet gp IIIa. The gp IIb-IIIa member of the integrin receptor family plays a role in the transmembrane linkage between its extracellular ligands and intracellular microfibers. Gp IIb-IIIa may thus contribute to normal synovial physiology and to the pathogenesis of chronic synovitis.

Immunological studies on patients with optic neuritis without evidence of multiple sclerosis.

We studied 6 patients with idiopathic optic neuritis (ON) after a mean follow-up period of 12 years. No evidence of multiple sclerosis (MS) was found on clinical grounds, nor by magnetic resonance imaging (MRI) or evoked responses. Levels of spontaneous proliferation, serum gamma-interferon (IFN-gamma) and lymphocyte MHC locus II antigen were similar in ON and in 6 patients with progressive MS, but different in healthy controls. This suggests that similar immunological conditions in vivo prevail after an idiopathic ON and in MS. A normal PHA-induced interleukin-2 receptor expression and lymphocyte proliferation but a low IFN-gamma secretion and MHC locus II antigen expression were observed. This suggests that an ON and MS defect is expressed after an interleukin-2 receptor ligand but before IFN-gamma secretion. Our findings also suggest that immunological factors do not explain the neuropathological confinement of the lesions in ON but the protective influence may rather be exerted by some as yet undefined genetic influence or may be explained by the different etiology of ON and MS.

Immune-inflammatory response in infected arthroplasties.

We studied the immunocytology of synovial fluid in purulent endoprosthetic infections using cell subtype-specific monoclonal antibodies in avidin-biotin-peroxidase complex staining. Two thirds of the monocytes were CD15-positive, whereas CD2-positive T lymphocytes only formed one third of all the mononuclear cells. The synovial fluid monocyte-activated T-cell ratio differed from findings in sterile inflammatory, reactive and rheumatoid arthritis.

PHA stimulation of peripheral blood lymphocytes in oral lichen planus. Abnormality localized between interleukin-2 receptor ligand formation and gamma-interferon secretion.

Peripheral blood T cell function in five oral lichen planus (OLP) patients and five healthy controls was assessed using different activation parameters. Staining with monoclonal antibodies against interleukin-2 receptor and MHC locus II coded Ia antigen, 3H-thymidine incorporation and gamma-interferon secretion were determined in phytohaemagglutinin (PHA) stimulated peripheral blood mononuclear cell cultures at days 0, 1, 3 and 5. The peripheral blood T cell subsets and spontaneous MHC locus II antigen expression were similar in OLP patients and in controls whereas the spontaneous lymphocyte proliferation was lower in OLP patients than in controls (p less than 0.01). This may reflect a slight in vivo preactivation and its effect on lymphocyte recirculation. The PHA-induced expression of IL-2 receptor and T cell proliferation were similar in both groups whereas gamma-interferon secretion and MHC locus II antigen expression were low in OLP patients compared with controls (p less than 0.01). The results suggest a defect in OLP T cell activation disclosed by in vitro PHA stimulation and localized between IL-2 receptor ligand binding and gamma-interferon secretion.

Synovial fluid lymphocytes in different subtypes of juvenile rheumatoid arthritis.

We studied the subsets of synovial fluid (SF) lymphocytes and their activation states in 4 subtypes of juvenile rheumatoid arthritis. The expression of lymphocyte differentiation antigens and activation markers (Ia and Tac) appeared to be similar in these subgroups. Tac + DNA-synthesizing T blasts represented, at most, 5% of all SF mononuclear cells. This finding was in clear contrast to the high proportion of Ia-positive SF mononuclear cells. There were no differences in Ia and Tac expression or DNA synthesis among the different juvenile rheumatoid arthritis subgroups. This finding suggests that the cell-mediated immune response may represent secondary features of the disease that are involved as a final common pathogenetic pathway.

An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis.

An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis is described. For this study expression of MHC locus II coded Ia antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein was analyzed using monoclonal antibodies in avidin-biotin-peroxidase complex staining combined with visualization of [3H]thymidine incorporation by autoradiography. Compared to spontaneous [3H]thymidine incorporation assay information is obtained at single cell level. In contrast to blast indexes calculated from MGG stained preparates, information on the expression of various functional cell surface structures as well as DNA synthesis is also obtained. Double-assay for lymphocyte phenotype and DNA synthesis by flow cytometry might be preferred to light microscopy, but the widespread use of immunoperoxidase staining and autoradiography may make this new kind of approach more easily available. Other advantages worth considering are the possibility of transporting, staining and counterstaining as microscope slides and the permanent nature of the documentation and morphological information obtained. In our experience, this method seems to be useful for studying resting peripheral blood lymphocytes as well as mitogen and antigen induced changes in the lymphocyte activation state.

Lymphocyte activation in oral lichen planus in situ.

The current study evaluated the activation of T-cell mediated cellular immune response in the oral lesions in lichen planus (OLP). Analysis was made of the ultramorphology, lymphocyte activation marker expression, DNA synthesis, and gamma-interferon production of the inflammatory cells in OLP lesions. According to these four different aspects of lymphocyte activation, only a minor fraction, 5% at the most, of all T-cells in situ were activated. This minor fraction, however, not the resting T-cells without these signs of activation, may decide the outcome of the local immune-inflammatory process in OLP. Of the inflammatory cells in situ, however, 81 +/- 5% were Ia positive. This finding may be the result of an Ia inducing capacity of the gamma-interferon produced locally by the activated T-cells.

Evaluation of lymphocyte activation in skin lesions of patients with mixed connective tissue disease and discoid lupus erythematodes.

Biopsy specimens from mixed connective tissue disease (MCTD) and discoid lupus erythematodes (DLE) skin lesions were stained with monoclonal antibodies to differentiation and activation antigens. In addition, the blast cells were studied by combining autoradiography with immunoperoxidase staining. In both disease conditions most of the inflammatory cells in situ were positive for T11 antigen, the CD4/CD8 ratio being low. Only a few of the cells were pan-B positive B cells. The expression of various activation antigens did not differ significantly between MCTD and DLE biopsy specimens; the number of T9, Tac, and 4F2 antigen carrying cells was relatively low, whereas Ia-positive cells were more numerous. 3H-Thymidine incorporating T blasts comprised less than 1% of all inflammatory cells. T4 and T8 marker-carrying blast cells were present in about equal proportions. These findings suggest that Ia antigen-expressing T cells are important from the pathogenetic point of view in both MCTD and DLE. Because the local proliferation of T cells was extremely low according to the lack of interleukin-2 receptor and OKT9 markers and 3H-thymidine incorporation, it seems probable that most of the T cells are recruited from the circulation to the site of the inflammation.