Cytokeratin Expression and Hyaluronic Acid Production in Cultures of Human Synovial Microvascular Endothelial Cells: Influence of Cytokines and Growth Factors

Objectives: To isolate and characterize human synovial endothelial cells and to determine the effects of cytokines and fibroblast growth factor on human synovial endothelial (HSE) cell hyaluronic acid production. Methods: Endothelial cells were isolated from primary cultures of human synovial cells by fluorescent activated cell sorting based on the incorporation of a fluorescent derivative of acetylated low-density lipoprotein (DiI-Ac-LDL). Identity of endothelial cells was confirmed by positive immunostaining for von Willebrand factor (vWf), cytokeratins, endoglin, and reactivity with the lectin ulex europeaus agglutinin (UEA). Hyaluronic acid production was measured by a radioligand-binding assay. Results: HSE cells were isolated and maintained in long-term culture. The identity of the cultured cells as endothelial was based on uniform uptake of a (DiI-Ac-LDL), immunoreactivity for vWf, and endoglin and the binding of the lectin UEA. In addition, small blood vessels in the synovium were stained selectively with anticytokeratin antibodies K4.62 (cytokeratin 19 specific) and K8.13 (reactive for cytokines 1, 5, 6, 7, 8, 10, 11, and 18). Isolated HSE cells also demonstrated immunoreactivity with these cytokeratin antibodies. The cytokeratins identified by the monoclonal antibody clone K8.13 demonstrated a diffuse, fibrillar staining pattern. The cytokeratin distribution revealed with monoclonal antibody K4.62 (cytokeratin 19) was also fibrillar; however, the majority of cells also demonstrated numerous punctate cytoplasmic vesicular structures. Treatment of HSE cells with interleukin-1α (IL-1α) or acidic fibroblast growth factor (aFGF), but not tumor necrosis factor (TNFα), dramatically reduced the vesicular structures staining with the K4.62 antibody. HSE cells produced hyaluronic acid (HA) at a constitutive rate of 200–800 ng/10⁵ cells/24 h, which could be upregulated when the cells were incubated with either IL-1α or aFGF. HA production was not significantly increased when HSE cells were incubated with TNFα, IL-4 or interferon-γ. Conclusions: Synovial microvascular endothelial cells produce and secrete HA and endothelial HA secretion is upregulated by IL-1 and aFGF. IL-1 and aFGF also reduce the number of vesicular-like structures immunoreactive with a monoclonal antibody to cytokeratin 19. These studies suggest that cytokine stimulation of local endothelial secretion and/or accumulation of HA may influence leukocyte adhesion to the synovial endothelium.     

Dynamics of interleukin (IL)-18 in serum, synovial fluid and synovial membrane in the patients with rheumatoid arthritis]

OBJECTIVES: IL-18 is a novel cytokine that plays an important role in the Th1 response. The aim of this study is to investigate the dynamics of IL-18 in serum, synovial fluid and synovial membrane in the patients with rheumatoid arthritis. MATERIALS AND METHODS: The serum, synovial fluid and synovial membrane were obtained from RA patients at operation. The levels of IL-18 in the serum and synovial fluid were measured by ELISA. We then examined the expression of IL-18 in synovial tissues using anti-human IL-18 monoclonal antibody in immunohistochemical study. RESULTS: The levels of IL-18 in serum and synovial fluid in RA patients were 193.7 +/- 109.7 pg/ml and 258.8 +/- 238.0 pg/ml, respectively. Compared with OA patients and normal volunteers, the level of IL-18 in RA patients was higher in both serum and synovial fluid. (P < 0.05) In synovial membrane, the cells positive for anti IL-18 antibody were confirmed not only in RA (n = 26) but also in OA (n = 7) patients. The positive cells were the synovial lining cells, macrophages, fibroblasts and endothelial cells. However, a large number of positive cells were demonstrated in synovial tissues in RA compared with OA patients.     

Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies.

The detection of early micrometastasis or disseminated single tumor cells poses a problem for conventional diagnosis procedures. Using a panel of monoclonal antibodies against cytokeratin and the 17-1A epithelial antigen we identified immunocytochemically tumor cells in bone marrow of patients with breast cancer (n = 155) and colorectal cancer (n = 57) at the time of surgery of the primary tumor. Monoclonal antibody CK2, recognizing the human cytokeratin component 18 in simple epithelia, appeared to be the most suitable reagent because of its negative reaction with bone marrow samples of the noncarcinoma patients (n = 75). Its specificity was further demonstrated in a double-marker staining procedure using an anti-leukocyte common antigen monoclonal antibody (T200) as counterstain. A comparative analysis showed that immunocytology was clearly superior to conventional cytology (n = 212) and histology (n = 39). In 9.5-20.5% of patients without distant metastasis, tumor cells could be detected in bone marrow. We found a significant correlation between tumor cells in bone marrow and conventional risk factors, such as distant metastasis or lymph node involvement. In a first approach toward immunotherapy we demonstrated in 3 patients that infused monoclonal antibody 17-1A can label single tumor cells in bone marrow in vivo. We then used this approach to follow up 7 patients undergoing 17-1A therapy in an adjuvant clinical trial.     

The development of the intrahepatic bile ducts in man: a keratin-immunohistochemical study

The development of the intrahepatic bile ducts in man was studied using an immunohistochemical technique on 56 liver specimens ranging in age from 6 weeks of gestation to 8 months after birth. On paraffin sections, two monoclonal anticytokeratin antibodies (CAM 5.2 and KL-1) that normally stain both hepatocytes and bile duct cells and two polyclonal anticytokeratin antisera that in normal adult liver stain bile ducts only were applied. For immunohistochemical staining of cryostat sections (only available from 14 weeks of gestation on), four monoclonal antibodies specifically directed against individual cytokeratin polypeptides 7,8, 18 and 19 were used. Adult human hepatocytes contain cytokeratin 8 and 18 whereas bile duct cells also express cytokeratin 7 and 19 in addition to cytokeratin 8 and 18. On paraffin sections, primitive hepatocytes were stained with monoclonal antibodies CAM 5.2 and KL-1 from 6 weeks of gestation on. On cryostat sections, they were positive with monoclonal antibodies anticytokeratin 8 and 18 from the earliest time point examined (14 weeks). From 9 weeks of gestation on, portal vein branches were surrounded by a layer of cells showing a stronger positive reaction with monoclonal antibodies CAM 5.2 and KL-1 on paraffin sections and with monoclonal antibodies anti-cytokeratin 8 and 18 on cryostat sections (only available from 14 weeks on). This layer, referred to as the ductal plate, first appeared around large portal vein branches close to the hilum and subsequently around more peripheral branches. The duct plates became duplicated over variably long segments of their perimeter, lumina appeared and tubular structures were formed. The latter gradually became incorporated in the connective tissue surrounding the portal vein, resulting in the appearance of individualized bile ducts. Ductal plate cells were stained by both polyclonal anticytokeratin antisera on paraffin sections. On cryostat sections (available from 14 weeks of gestation on), they were immunoreactive for cytokeratin 19 but negative with the monoclonal antibody directed against cytokeratin 7 until 20 weeks of gestation. From then on, weakly positive staining for cytokeratin 7 was detected, but staining intensity subsequently increased and reached the level observed in adult liver at 1 month after birth. At birth, the smallest branches of the portal vein were still surrounded by a discontinuous ductal plate. We conclude that intrahepatic bile duct cells develop from hepatocytes around branches of the portal vein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fibroblast growth factors released by wounded endothelial cells stimulate proliferation of synovial cells.

We demonstrated that vascular endothelial cells mechanically wounded by scraping from the substratum were able to release growth promoting factors for synovial cells as well as for endothelial cells. The wounded endothelial cell conditioned medium stimulated the proliferation of synovial cells derived from different human donors dose dependently and induced transit of growth arrested synovial cells (predominant in the G0 and G1 phases), through the S phase and into the G2 and M phases. When the wounded endothelial cell conditioned medium was applied to heparin-sepharose columns, mitogenic activity was eluted with 2.0 M NaCl. The mitogenic activity in wounded endothelial cell conditioned medium, which was heat sensitive, was inhibited by protamine sulfate and a specific mouse monoclonal antibody against basic fibroblast growth factor (bFGF). Our results are evidence that the wounded endothelial cell conditioned medium contained growth promoting factors for synovial cells, including bFGF. We also showed the presence of bFGF in the endothelial cells of the small blood vessels and stromal synovial fibroblast-like cells in patients with rheumatoid arthritis (RA). Our results suggest that the endothelial cells in the luxuriant capillaries in the synovial tissues from patients with RA may have a critical role in the stimulation of neighboring synovial cell proliferation, resulting in pronounced synovial hyperplasia.     

Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis,spondyloarthropathies and osteoarthritis and normal controls

OBJECTIVES: To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis. RESULTS: Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration. CONCLUSIONS: OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.     

Dendritic cells and high endothelial venules in the rheumatoid synovial membrane

Since dendritic cells are believed to play a crucial role in the pathogenesis of rheumatoid arthritis (RA) we studied the microenvironmental relationship of these cells with endothelial cells, lymphocytes and macrophages in the rheumatoid synovial membrane. With the monoclonal antibodies OKIa (MHC Class II determinants), RFD1 and L25 (both specific for "active" human dendritic cells) we identified large numbers of dendritic cells. With the monoclonal antibody HECA 452 [specific for a putative adhesion molecule notably present on high endothelial venules (HEV)], a subset of dendritic cells could be detected. HECA-452 positive dendritic cells were found in 2 basic patterns: (1) associated with small lymphoid cell clusters in the neighborhood of vessels with flat, HECA-452 negative endothelium, (2) at the periphery of dense organoid lymphoid infiltrates, surrounding HECA-452 positive HEV-like vessels. Our data suggest that the influx of HECA-452, L25, RFD1 and MHC Class II positive dendritic cells is an early event in the development of the inflammatory infiltrate found in the rheumatoid synovial membrane. The formation of organoid lymphoplasmacellular infiltrates with high endothelial venules would be secondary to this event.     

Elevation of Anti-cytokeratin 19 Antibody in Sera of the Patients With Idiopathic Pulmonary Fibrosis and Pulmonary Fibrosis Associated With Collagen Vascular Disorders

It has been suggested that cytokeratin 19 is expressed in regenerated bronchoepithelial cells in patients with pulmonary fibrosis, and serum cytokeratin 19 fragment is elevated in patients with pulmonary fibrosis. We hypothesized that serum antibodies to cytokeratin 19 may be formed in patients with pulmonary fibrosis. To prove the existence of anti-cytokeratin 19 antibodies in patients' sera, human recombinant cytokeratin 19 was stained with patients' sera by a Western immunoblot. Then, we tried to establish an enzyme-linked immunosorbent assay to quantitate anti-cytokeratin 19 antibody in the sera of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis associated with collagen vascular disorders (PF-CVD). We demonstrated the anti-cytokeratin 19 antibody in patient' sera by a Western immunoblot. In patients with IPF and PF-CVD, significantly high anti-cytokeratin 19 antibody was demonstrated compared with normal volunteers, patients with chronic bronchitis, and patients with pneumonia. These results suggest that anti-cytokeratin 19 antibody may have played a role in the process of lung injury in pulmonary fibrosis. Accepted for publication: 27 May 1999     

Immunohistochemical localization of somatostatin receptor sst2A in human rheumatoid synovium

OBJECTIVE: To identify the somatostatin receptor-expressing cells in rheumatoid synovium using a recently developed antiserum directed against the somatostatin receptor subtype 2A (sst2A). METHODS: We carried out immunohistochemical studies of synovial biopsies from 7 patients with rheumatoid arthritis (RA) and one non-RA patient, using a rabbit polyclonal antiserum directed against sst2A and monoclonal antibodies directed against phenotypic markers. RESULTS: SSt2A was expressed by the endothelial cells of the synovial venules but also by a subset of synovial macrophages. CONCLUSION: The identification of somatostatin receptors on macrophages, which are thought to be important effector cells in RA, may offer mechanistic insights into the potential therapeutic effect of somatostatin (analogs) in RA.     

Reversible opening of the blood-brain barrier by anti-bacterial antibodies.

The leukocyte adhesion molecule CR3 (CD11b/CD18, Mac-1) promotes leukocyte transmigration into tissues by engaging an unknown cognate ligand on the surface of vascular endothelial cells. Filamentous hemagglutinin (FHA), an adhesin of the bacterium Bordetella pertussis, binds to CR3. We hypothesized that FHA mimics the native ligand for the CR3 integrin on endothelial cells and predicted that anti-FHA antibodies should bind to endothelial cells, interfere with leukocyte recruitment, and induce endothelial permeability. Anti-FHA monoclonal antibodies bound to cerebral microvessels in sections from human brain and upon intravenous injection into rabbits. Antibody binding correlated with the ability to recognize two polypeptides in extracts of human cerebral vessels that were also bound by CD18. In vivo, antibody binding not only interfered with transmigration of leukocytes into cerebrospinal fluid but also induced a dose-dependent reversible increase in blood-brain barrier permeability sufficient to improve delivery of intravenously administered therapeutic agents to brain parenchyma.     

ADHESION OF RHEUMATOID LYMPHOCYTES TO MUCOSAL ENDOTHELIUM: THE GUT REVISITED

By a study of the adhesion of rheumatoid mononuclear cells,we have sought to clarify the homing properties and originsof cells likely to be involved in the pathogenesis of this disease.The adhesion of mononuclear cells from patients with rheumatoidarthritis (RA) was enumerated by an in vitro adherence assayusing frozen sections of endothelium-containing gut lamina propria(EGLP) from porcine small intestine. Preliminary studies verifiedthe involvement of known adhesion molecules by inhibition assaysusing monoclonal antibodies Meca-367, Mel-14 and Hermes-3. Twenty-fivepaired samples of peripheral blood (PB) and synovial fluid (SF)were studied, plus six from synovial membrane (SM) and eightfrom patients with other diseases. There was a significantlygreater degree of adherence to EGLP by SF cells than PB (meanadherence 266 ± 22 cells/mm², compared to 136 ±13 cells/mm², respectively, the majority of which were CD8+cells; P = 0.02, Mann-Whitney U-test for 25 paired samples).The results of the monoclonal antibody inhibition assays werein keeping with the involvement of homing receptors to gut endotheliumin our assay system. Synovial fluid lymphocytes from RA patientsexhibited adhesion properties more in keeping with lymphocytesof mucosal than of lymph node origin. Synovial membrane lymphocytes,by contrast, showed poor adherence to endothelium-containinglamina propria. The gut, as an immune lymphoid organ, may thusplay a contributory role in this disease, possibly through thepathological seeding of cells into the synovial space. KEY WORDS: Rheumatoid arthritis, Adhesion, MALT     

Increased IgA antibodies to cytokeratins in the spondyloarthropathies.

OBJECTIVES--Increased levels of IgA antibodies to cytokeratin-18 (CK-18) and epidermal keratins (EpK) in the sera of patients with rheumatoid arthritis (RA) have been demonstrated previously. In the present study investigations were carried out to determine whether levels of these autoantibodies were also raised in the spondyloarthropathies, and whether there was any association with particular disease manifestations. METHODS--Using specific enzyme linked immunosorbent assays (ELISA) measurements were taken of IgA, IgG and IgM antibodies to EpK and to CK-18 in the sera of patients with psoriatic arthropathy, ankylosing spondylitis (AS), Reiter's syndrome, psoriasis and in normal subjects. RESULTS--IgA antibodies to both EpK and CK-18 were significantly increased in sera from patients with psoriasis and psoriatic arthropathy but not in the sera from the patients with AS or Reiter's syndrome, or in the controls. In psoriatic arthritis, however, these levels were significantly higher only in those patients with peripheral joint disease and not in those with axial arthritis alone. There was no significant increase in antibody levels in patients with AS or Reiter's syndrome. There were no differences in the levels of IgG or IgM antibodies to CK-18 or EpK between the patient groups and controls. CONCLUSIONS--Raised levels of IgA antibodies to CK-18 and EpK in psoriatic arthropathy and psoriasis probably reflect exposure of intracellular cytokeratin antigens to the immune system after damage to cytokeratin containing cells, and suggests a common pathogenic mechanism in these conditions which involves production of cytokeratin autoantibodies. In patients with psoriatic arthropathy, such a mechanism appears only to be operating in patients with peripheral joint involvement and not in those with axial arthritis.     

THE ROLE OF IMMUNOPEROXIDASE TECHNIQUES ON PARAFFIN EMBEDDED TISSUE IN DETERMINING THE HISTOGENESIS OF UNDIFFERENTIATED THYROID NEOPLASMS

Twenty cases of undifferentiated thyroid tumours were reviewed histologically. In seven cases the histogenesis was difficult to determine using morphological criteria. Immunohistochemical staining with a panel of antibodies to lymphoid and epithelial cells, including monoclonal antibodies directed against the leucocyte common antigen, cytokeratin, and epithelial membrane antigen confirmed that four of these cases were lymphomas and that one was a medullary carcinoma. In the remaining two cases immunohistochemistry was unhelpful. In the thirteen histologically typical tumours, the immunohistochemical profile was in keeping with their histogenesis as determined by morphological criteria. Immunohistochemical staining with a panel of selected antibodies allows the reliable diagnosis of undifferentiated thyroid neoplasms, when this cannot be reached using routine histological techniques.     

Phenotypic characterization of 3H-thymidine incorporating cells in rheumatoid arthritis synovial membrane

Summary Tritiated-thymidine incorporating cells in synovial tissue samples from ten patients with definite or classic rheumatoid arthritis (RA) were studied by combining two techniques. Tritiated-thymidine-labelled cells were seen in autoradiography and simultaneously the subtype of them was determined with immunoperoxidase staining using monoclonal antibodies. Tritiated-thymidine-labelled cells comprised 0.8±0.4% of all the inflammatory cells in RA synovial membrane. Of all ³H-thymidine-labelled cells 34±17% were positive for OKT8 and 19±8% for OKT4 monoclonal antibodies. OKM1-positive cells comprised 7±3% of all ³H-thymidine labelled cells, whereas only a few (3±4%) of them were positive for pan-B monoclonal antibody. This study emphasizes the importance of activated OKT8 lymphocytes in RA synovial membrane.     

CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A alpha chain of fibrinogen.

Fibrinogen and fibrin serve as adhesive substrates for a variety of cells including platelets, endothelial cells, and leukocytes. Previously, we identified the C terminus of the gamma chain of fibrinogen as the region of the fibrinogen molecule that contains a ligand for CD11b/CD18 (complement receptor 3) on phorbol ester-stimulated polymorphonuclear leukocytes. In contrast, we report here that neutrophils stimulated with tumor necrosis factor adhere to fibrinogen-coated surfaces, but not to human serum albumin-coated surfaces, via the integrin CD11c/CD18 (p150/95). Monoclonal antibodies LeuM5 and 3.9, which are directed against the alpha subunit of CD11c/CD18, but not monoclonal antibodies OKM10 and OKM1, which are directed against the alpha subunit of CD11b/CD18, inhibit the adhesion of tumor necrosis factor-stimulated neutrophils to fibrinogen-coated surfaces. To identify the site on fibrinogen recognized by CD11c/CD18, we have examined the adhesion of tumor necrosis factor-stimulated neutrophils to surfaces coated with various fibrinogen fragments. Stimulated neutrophils adhere to surfaces coated with the N-terminal disulfide knot fragment of fibrinogen or fibrinogen fragment E. Moreover, peptides containing the sequence Gly-Pro-Arg (which corresponds to amino acids 17-19 of the N-terminal region of the A alpha chain of fibrinogen), and monoclonal antibody LeuM5, block tumor necrosis factor-stimulated neutrophil adhesion to fibrinogen and to the N-terminal disulfide knot fragment of fibrinogen. Thus, CD11c/CD18 on tumor necrosis factor-stimulated neutrophils functions as a fibrinogen receptor that recognizes the sequence Gly-Pro-Arg in the N-terminal domain of the A alpha chain of fibrinogen.     

Monoclonal autoantibodies to cardiolipin derived from SLE mice.

The objective of this study was to clarify the specificity of anticardiolipin antibodies (aCL). Eighteen monoclonal hybridoma aCL from systemic lupus erythematosus (SLE)-prone MRL/Mp-lpr/lpr mice were established, and the reactivity of monoclonal aCL to phospholipids, DNA, nuclei of human epithelial cells, platelets, vascular endothelial cells, heparin, protein C and thrombomodulin was examined. All the 18 monoclonal aCL reacted with phosphatidylserine and some showed reactivity to phosphatidylinositol and phosphatidylcholine. Six of 16 monoclonal aCL were demonstrated to have the property of lupus anticoagulant. Monoclonal aCL were classified into three categories, in terms of DNA-binding specificity. Ten of 18 aCL had characteristics of antinuclear antibodies. Six of 11 aCL reacted with platelets. Three of 18 aCL were bound to vascular endothelial cells and to heparin. No monoclonal aCL reacted with protein C or thrombomodulin. Therefore, the conclusion was made that monoclonal aCL from SLE mice showed a polyspecific nature.     

Characterization of endothelial cells in murine long-term marrow culture. Implication for hemopoietic regulation.

Establishing the presence of various cell types in long-term marrow culture (LTMC) has an important bearing on understanding the regulation of stromal cell-related hemopoiesis. Controversy has surrounded the identity of the very large cells in murine LTMC; they have been given a variety of designations, including blanket cells. Using dual immunogold labeling with a recently derived monoclonal antibody, H513E3, and anti-human factor VIII antibodies, we have conclusively located endothelial cells in LTMC. Endothelial cells are relatively large, with thinly spread cytoplasm, and they overlie macrophages, granulocytes, and other less differentiated developing hemopoietic cells. Previously, demonstration of endothelial cells in LTMC had been difficult due to lack of specific markers in the mouse. We have demonstrated that LTMC endothelial cells have the exact location and ultrastructural characteristics as the previously described blanket cells. We propose that previous designations should not be continued and that these cells should be referred to as endothelial cells. The known functions of endothelial cells now become relevant to the understanding of stromal regulation of hemopoiesis.     

Analysis of cartilage oligomeric matrix protein (COMP) in synovial fibroblasts and synovial fluids

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes.      

Induction and enhancement by monocytes of antibody-induced modulation of a variety of human lymphoid cell surface antigens

We have previously reported that the addition of monocytes results in enhanced modulation of the T65 antigen when normal or leukemic lymphoid cells were cultured in vitro with the T101 monoclonal antibody. In the present investigation, we extend these findings to demonstrate that monocyte-enhanced modulation is a phenomenon that occurs with a variety of T and B lymphoid antigens identified by murine monoclonal antibodies. Two patterns of monocyte-enhanced modulation were observed: (1) augmentation by monocytes of existing antigen modulation by the T101 and anti-Leu-4 antibodies, and (2) induction by monocytes of previously unrecognized modulation with the anti-Leu-2 and anti-Leu-9 antibodies. Enhancement of modulation by monocytes was also detected with antibodies to surface IgM and HLA-DR antigens. Antigen modulation on lymphoid cell lines appeared to be more variable than on fresh cells, with or without monocytes. Monocyte-enhanced antigen modulation was not demonstrated with two monoclonal antibodies against solid tumors. Monocyte-enhanced modulation was shown to be dependent upon the Fc portion of the antibody, but independent of proteolytic or oxidative compounds released by monocytes. These findings indicate that the results obtained during in vitro studies of antigen modulation may vary with the source of cells and the extent to which monocytic cells are present. In addition, these findings suggest an enhanced role for Fc receptor-bearing cells of monocytic origin in antigen modulation following in vivo administration of monoclonal antibodies.     

Localization of tumor necrosis factor receptors in the synovial tissue and cartilage-pannus junction in patients with rheumatoid arthritis. Implications for local actions of tumor necrosis factor alpha.

OBJECTIVE. We have previously described the location of tumor necrosis factor alpha (TNF alpha)-producing cells in synovial tissue and cartilage-pannus junction in rheumatoid arthritis (RA). To further understand the local actions of TNF alpha, we investigated the expression of TNF receptors (TNF-R) on cells in the same compartments in patients with RA. METHODS. The expression of both p55 TNF-R and p75 TNF-R was determined using alkaline phosphatase-conjugated mouse anti-alkaline phosphatase (APAAP) and double immunofluorescence staining techniques with monoclonal antibodies. RESULTS. In RA synovial membrane, both p55 TNF-R and p75 TNF-R were detectable in up to 90% of the cells in the lining layer, and were demonstrated on cells in deeper layers of the membrane, including vascular endothelial cells. Cells in lymphoid aggregates expressed both TNF-R, but with a predominant expression of p75 receptor. At the cartilage-pannus junction, the majority of pannus cells, especially those invading cartilage, expressed both the p55 and the p75 TNF-R. Sequential section and double immunofluorescence staining showed that the TNF-R-expressing cells were in the vicinity of TNF alpha-containing cells, and some TNF alpha-containing cells also expressed TNF-R. TNF-R-expressing cells were also detected in osteoarthritic and normal synovial tissue, but in smaller numbers and at a lower intensity. CONCLUSION. These results provide histologic evidence that both p55 TNF-R and p75 TNF-R are expressed by a variety of cell types in RA synovial tissue, reflecting the fact that a wide range of cells are potential targets for TNF alpha in this tissue. This study further supports the hypothesis that TNF alpha plays a major role in the pathogenesis of RA.