Structural basis for autoantibody recognition of phosphatidylserine-beta 2 glycoprotein I and apoptotic cells.

Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by beta 2 glycoprotein I (beta 2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-beta 2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.

Increased CD95/Fas-induced apoptosis of HIV-specific CD8(+) T cells.

Why HIV-specific CD8(+) T cells ultimately fail to clear or control HIV infection is not known. We show here that HIV-specific CD8(+) T cells exhibit increased sensitivity to CD95/Fas-induced apoptosis. This apoptosis is 3-fold higher compared to CMV-specific CD8(+) T cells from the same patients. HIV-specific CD8(+) T cells express the CD45RA(-)CD62L(-) but lack the CD45RA(+)CD62L(-) T cell effector memory (T(EM)) phenotype. This skewing is not found in CMV- and EBV-specific CD8(+) T cells in HIV-infected individuals. CD95/Fas-induced apoptosis is much higher in the CD45RA(-)CD62L(-) T(EM) cells. However, cytotoxicity and IFNgamma production by HIV-specific CD8(+) T cells is not impaired. Our data suggest that the survival and differentiation of HIV-specific CD8(+) T cells may be compromised by CD95/Fas apoptosis induced by FasL-expressing HIV-infected cells.

An early oxygen-dependent step is required for dexamethasone-induced apoptosis of immature mouse thymocytes.

The roles of oxygen and reactive oxygen intermediates in apoptosis are unclear at present. Although oxygen and reactive oxygen intermediates are not required for the execution of apoptosis, oxygen may be involved in at least some forms of apoptosis. In this study we show that dexamethasone (Dex)-induced apoptosis of immature mouse thymocytes is completely inhibited by hypoxic culture. In contrast, anti-CD95 thymocyte apoptosis is unaffected by hypoxia, indicating the existence of two forms of thymocyte apoptosis: an oxygen-dependent pathway (Dex induced) and an oxygen-independent pathway (anti-CD95 induced). Furthermore, hypoxia inhibited mitochondrial permeability transition (PT) in Dex-treated, but not in anti-CD95-treated, thymocytes, suggesting that the oxygen-sensitive step is upstream of mitochondria. Both Dex- and anti-CD95-induced PT and apoptosis were dependent on activation of IL-converting enzyme-like protease, as PT and apoptosis were inhibited by preincubation with Cbz-Val-Ala-Asp-fluoromethyl ketone, an irreversible inhibitor of IL-converting enzyme-like proteases. In addition, hypoxia inhibited the activation by Dex of caspase-3 (CPP32)-like proteases. Our data show that the private signaling pathways of Dex (oxygen dependent) and anti-CD95 (oxygen independent) both converge upstream of mitochondrial changes. The oxygen-dependent step in Dex-induced apoptosis lies upstream of caspase-3-like protease activation. Our observations support a model of apoptosis signaling in which independent pathways (oxygen dependent and oxygen independent) particular to each stimuli converge at a central point in the apoptotic cascade.

Imaging of apoptosis (programmed cell death) with 99mTc annexin V.

UNLABELLED: Apoptosis (programmed cell death) is a critical element in normal physiology and in many disease processes. Phosphatidylserine (PS), one component of cell membrane phospholipids, is normally confined to the inner leaflet of the plasma membrane. Early in the course of apoptosis, this phospholipid is rapidly exposed on the cell's outer surface. Annexin V, an endogenous human protein, has a high affinity for membrane-bound PS. This protein has been labeled with fluorescein and has been used to detect apoptosis in vitro. We describe the use of radiolabeled annexin V to detect apoptosis in vivo. The results are compared to histologic and flow cytometric methods to identify cells and tissues undergoing apoptosis. METHODS: Annexin V was coupled to hydrazinonicotinamide (HYNIC) and radiolabeled with 99mTc. Bioreactivity of 99mTc-HYNIC annexin V was compared with fluorescein isothiocyanate (FITC)-labeled annexin V in cultures of Jurkat T-cell lymphoblasts and in ex vivo thymic cell suspensions undergoing apoptosis in response to different stimuli. In addition, the uptake of FITC annexin V and 99mTc-HYNIC annexin V was studied in heat-treated necrotic Jurkat T-cell cultures. In vivo localization of annexin V was studied in Balb/c mice injected with 99mTc-HYNIC annexin V before and after induction of Fas-mediated hepatocyte apoptosis with intravenously administered antiFas antibody. RESULTS: Membrane-bound radiolabeled annexin V activity linearly correlated to total fluorescence as observed by FITC annexin V flow cytometry in Jurkat T-cell cultures induced to undergo apoptosis in response to growth factor deprivation (N = 10, r2 = 0.987), antiFas antibody (N = 8, r2 = 0.836) and doxorubicin (N = 10, r2 = 0.804); and in ex vivo experiments on thymic cell suspensions with dexamethasone-induced apoptosis from Balb/c mice (N = 6, r2 = 0.989). Necrotic Jurkat T-cell cultures also demonstrated marked increases in radiopharmaceutical (4000-5000-fold) above control values. AntiFas antibody-treated Balb/c mice (N = 6) demonstrated a three-fold rise in hepatic uptake of annexin V (P < 0.0005) above control (N = 10), identified both by imaging and scintillation well counting. The increase in hepatic uptake in antiFas antibody-treated mice correlated to histologic evidence of fulminant hepatic apoptosis. CONCLUSION: These data suggest that 99mTc-HYNIC annexin V can be used to image apoptotic and necrotic cell death in vivo.

In situ staining for poly(ADP-ribose) polymerase activity using an NAD analogue.

Poly(ADP-ribose) polymerase (PARP) is a highly abundant nuclear enzyme which metabolizes NAD, in response to DNA strand breakage, to produce chains of poly(ADP-ribose) attached to nuclear proteins. PARP activation has been implicated in ischemia/reperfusion injury, but its biological significance is not fully understood. We have modified an existing in situ method for detection of PARP activity by using an NAD analogue in which adenine is modified by an "etheno" (vinyl) bridge. Etheno-NAD serves as a PARP substrate in an initial enzymatic reaction; a specific antibody to ethenoadenosine is then used in an immunohistochemical reaction to detect the production of modified poly(ADP-ribose). The method produces strong and specific labeling of nuclei in which PARP has been activated, i.e., those in which DNA strand breaks have been produced, and the results can be analyzed by microscopy, flow cytometry, or colorimetry. The method is applicable to cultured cells in several formats and to frozen tissue sections. The particular characteristics of the new method may assist in future in situ studies of PARP activation.

In vivo detection and imaging of phosphatidylserine expression during programmed cell death.

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.

Interleukin-1 beta converting enzyme-like protease involvement in Fas-induced and activation-induced peripheral blood T cell apoptosis in HIV infection. TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in HIV infection.

Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)-induced and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals. In this study, we extend and confirm these observations by using an inhibitor of interleukin-1 beta converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4(+) and CD8+ T cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)- induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this AICD is mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different roles in the pathogenesis of HIV infection.

HIV type 1 Tat protein enhances activation-but not Fas (CD95)-induced peripheral blood T cell apoptosis in healthy individuals.

T cell apoptosis may play an important role in the depletion and functional defects of T cells in HIV disease. A number of investigators have shown that peripheral blood T cells in HIV disease undergo spontaneous and activation-induced apoptosis. We found recently that peripheral blood T cells from HIV+ individuals undergo apoptosis when stimulated through Fas. Also, a number of investigators have shown that Tat protein from HIV-1 can increase spontaneous and activation-induced apoptosis. In the present study we examined the effect of HIV type 1 Tat protein on spontaneous, activation-induced and Fas-induced apoptosis of peripheral blood T cells from HIV- individuals. We find that Tat protein has no effect on spontaneous apoptosis but does enhance activation-induced apoptosis of both CD4+ and CD8+ T cells. Tat, however, failed to enhance Fas-induced apoptosis of CD4+ and CD8+ T cells. Examining the mechanisms by which Tat induces apoptosis, we found that inhibitors of reactive oxygen intermediate (ROI) generation or neutralizers of ROI, such as rotenone, a potent inhibitor of mitochondrial complex I of the respiratory chain, and 3,3,5,5-tetramethylpyrroline N-oxide (TMPO), an electron spin trap, could both enhance the spontaneous apoptosis induced by Tat. This enhancement of Tat-induced apoptosis by rotenone and TMPO was independent of ICE activation as it could not be inhibited by the tripeptide z-VAD-fmk, an irreversible inhibitor of ICE/ced-3 protease homologs. These findings suggest that Tat induced enhancement of activation-induced cell death may involve complex mechanisms, some of which are ROI independent. These results indicate that a HIV-specific mechanism other than Tat is responsible for the previously observed increased susceptibility of peripheral blood T cells from HIV-infected individuals to undergo apoptosis in response to Fas stimulation.

Quantitative analysis of apoptotic cell death using proton nuclear magnetic resonance spectroscopy.

Quantification of apoptotic cell death in vivo has become an important area of investigation in patients with acute lymphoblastic leukemia (ALL). We have devised a noninvasive analytical method to estimate the percentage of apoptotic lymphoblasts in doxorubicin-treated Jurkat T-cell ALL cultures, using proton nuclear magnetic resonance spectroscopy (1H NMR). We have found that the ratio of the methylene (CH2) resonance (at 1.3 ppm) to the methyl (CH3) resonance (at 0.9 ppm) signal intensity, as observed by 1H NMR, is directly proportional to the percentage of apoptotic lymphoblasts in vitro. The correlation between the CH2/CH3 signal intensity ratio and the percentage of apoptotic lymphoblasts was optimal 24 to 28 hours after doxorubicin treatment (r2 = .947, N = 27 samples). There was also a direct temporal relationship between an increase in the CH2/CH3 signal intensity ratio and the onset of apoptosis as detected by nuclear morphologic analysis, fluorescein-annexin V flow cytometry, and DNA gel electrophoresis. Thin-layer chromatography confirmed that a dynamic and/or compositional change of the plasma membrane, rather than increases in lipase activity or fatty acid production, appears to account for the increase in the CH2/CH3 signal intensity ratio during apoptosis. 1H NMR may have clinical utility for the early noninvasive assessment of chemotherapeutic efficacy in patients with ALL.

Activation-induced peripheral blood T cell apoptosis is Fas independent in HIV-infected individuals.

T cell apoptosis has been proposed as an important contributor to the functional defects and depletion of T cells in HIV-infected individuals. However, the mechanisms involved in this apoptosis have not been elucidated. We recently showed that peripheral blood T cells from HIV-infected individuals are especially susceptible to Fas antigen-induced apoptosis. In this study we examine the role of Fas, CTLA-4, tumor necrosis factor (TNF) receptors (TNFR) and CD30, receptors known to be involved in T cell activation-induced cell death (AICD), in the spontaneous and activation (anti-CD3)-induced apoptosis of peripheral blood T cells from asymptomatic HIV-infected individuals. We report here that spontaneous and activation-induced T cell apoptosis cannot be inhibited by reagents that block interactions of Fas, CTLA-4, p55 and p75 TNFR and CD30 with their respective ligands. We also show that IL-12, IFN-gamma, IL-4 and IL-10 cannot modify spontaneous, activation- and anti-Fas-induced apoptosis. Anti-Fas preferentially induced CD4+ T cell apoptosis whereas AICD induced apoptosis equally in CD4+ and CD8+ T cells. We conclude that T cell AICD in HIV infection is not mediated by Fas, thus indicating that Fas-induced and activation-induced T cell apoptosis are independent mechanisms of apoptosis which may play different roles in the pathogenesis of HIV infection.

Interleukin-10 inhibition of the progression of established collagen-induced arthritis.

OBJECTIVE: Interleukin-10 (IL-10) is a potent inhibitor of the proinflammatory cytokines, including tumor necrosis factor alpha and IL-1, which are considered important in the pathogenesis of rheumatoid arthritis (RA). The study was undertaken to establish whether IL-10 can ameliorate arthritis in the collagen-induced arthritis (CIA) model of RA. METHODS: DBA/1 mice were immunized with bovine type II collagen in adjuvant, and treated daily after disease onset with recombinant murine IL-10 or with saline as a control. Mice were monitored for paw swelling and clinical score. Histologic analysis was also performed. RESULTS: IL-10 treatment of established CIA inhibited paw swelling (P < 0.0001), as well as disease progression as defined by clinical score (P < 0.0002). Cartilage destruction, as assessed histologically, was reduced in IL-10-treated mice compared with controls (P < 0.01). CONCLUSION: IL-10 suppresses established CIA, probably by inhibiting proinflammatory cytokine production. Our results, taken together with previously reported findings, indicate a potential therapeutic role for IL-10 in RA.

Are CD4+ Th1 cells pro-inflammatory or anti-inflammatory? The ratio of IL-10 to IFN-gamma or IL-2 determines their function.

Human CD4+ T cells have, like their murine counterparts, been classified on the basis of their cytokine profile. Th1 cells produce IL-2 and IFN-gamma, but little or no IL-4. Th2 cells produce IL-4 but not IFN-gamma or IL-2, and Th0 produce IL-2, IL-4 and IFN-gamma. As IL-2 is the most potent T cell growth factor and IFN-gamma is the strongest activator of macrophages it is not surprising that CD4+ Th1 cells are considered to be pro-inflammatory. However, unlike results in the mouse, where IL-10 is only produced by Th2 cells, human IL-10 is produced by Th0, Th1 and Th2 cells. Hence some human Th1 cells are capable of producing both pro-inflammatory (IL-2, IFN-gamma) and anti-inflammatory (IL-10) cytokines, therefore the function of these cells may not be accurately encapsulated by the 'Th1' terminology. We thus investigated the correlation of cytokine production and function in human CD4+ Th1 clones. Cytokine production (IL-2, IFN-gamma, IL-10) was measured in supernatants by ELISA after stimulation with solid-phase anti-CD3. The capacity of these supernatants to activate or inhibit T cell proliferation or LPS induced TNF-alpha production by monocytes was assessed. The ratio of IL-2/IL-10 or IFN-gamma/IL-10 was of critical importance in determining the function of the supernatants. The inhibitory effects were verified to be due to IL-10, as they were neutralized by anti-IL-10 mAb.(ABSTRACT TRUNCATED AT 250 WORDS)

High level of interleukin-10 production by the activated T cell population within the rheumatoid synovial membrane.

OBJECTIVE: To characterize the cytokine profile of the activated T cell population derived from the synovial membrane of rheumatoid arthritis (RA) patients. METHODS: Interleukin-2 (IL-2) was used to select for in vivo-activated T cells from the synovial membrane of 2 patients with RA, and the cells were cloned nonspecifically. The cytokine production profile of these clones was compared with that of clones derived from peripheral blood monocytes (PBM) by stimulating all clones for 24 hours with immobilized anti-CD3 (coated at 10 micrograms/ml) or phorbol-12-myristate-13-acetate (10 ng/ml) plus soluble anti-CD3 (1 microgram/ml). Interferon-gamma (IFN gamma), IL-4, and IL-10, the cytokines that discriminate between Th1 and Th2 cells and are involved in immunoregulation, were assayed by enzyme-linked immunosorbent assay. RESULTS: There was a difference in the cytokines produced by the clones derived from the rheumatoid membranes compared with clones derived from the periphery. Clones derived from both membranes and PBM were mostly IFN gamma-producers, i.e., either a Th0 or a Th1 profile. There was a high proportion of IFN gamma/high IL-10-producing cells derived from the joint, but not from the periphery. Of clones derived from the synovial membrane of each of 2 RA patients, 100% and 50% produced both 1-10 ng/ml IFN gamma and > 7 ng/ml IL-10, compared with < 7% of clones derived from normal or RA peripheral blood. In addition, when autologous membrane and PBM were compared, the mean concentration of IL-10 produced by the clones derived from the synovial membrane sample was significantly different from those produced by clones derived from peripheral blood (P < 0.02). CONCLUSION: The cytokine profile of the T cell clones that were obtained from the RA joint after expansion with IL-2 is distinct from that of the T cells that are predominant in PBM. This supports the concept that the T cells subsets that accumulate in the joint are not a random sample. The high level of IL-10 production by clones derived from the synovium suggests that this cytokine may be a major contributor to the endogenous immunosuppression that occurs in RA.

Fas antigen stimulation induces marked apoptosis of T lymphocytes in human immunodeficiency virus-infected individuals.

Apoptosis (programmed cell death) of T lymphocytes has been proposed as a mechanism which plays an important role in the pathogenesis of human immunodeficiency virus (HIV) disease. Activation of Fas (CD95) can either result in costimulation of proliferation and cytokine production or in the induction of apoptosis of T lymphocytes. This raises the possibility that Fas is involved in the observed T cell apoptosis during HIV disease. In this report we show that peripheral blood CD4+ and CD8+ T lymphocytes from HIV-infected individuals undergo apoptosis in vitro in response to antibody stimulation (cross-linking) of Fas at a much higher frequency than from uninfected controls. This anti-Fas-induced T cell apoptosis is markedly higher than spontaneous T cell apoptosis in HIV-infected individuals. Antibodies against other members of the tumor necrosis factor (TNF)/nerve growth factor receptor family such as CD27, CD30, CD40, 4-1BB, p55 TNF receptor, p75 TNF receptor, and TNF receptor-related protein did not result in any increase of T cell apoptosis above that spontaneously observed in HIV+ individuals. Anti-Fas-induced apoptosis was much higher in symptomatic HIV-infected individuals; and the magnitude of anti-Fas-induced CD4+ T cell apoptosis correlated inversely with peripheral blood CD4+ T cell absolute counts. Surface expression of Fas on T cells was also found to be higher in HIV-infected individuals. Resting and activated CD4+ and CD8+ T cells both underwent apoptosis in response to anti-Fas antibody. L-Selectin positive memory CD4+ T cells were especially susceptible to anti-Fas-induced apoptosis. These findings show that CD4+ and CD8+ T lymphocytes in HIV-infected individuals are primed in vivo to undergo apoptosis in response to Fas stimulation, suggesting that Fas signaling may be responsible for the T lymphocyte functional defects and depletion observed in HIV disease.

Human alpha beta T-cell receptor CD4-CD8 T-cell clones are predominantly Th0-like.

The cytokine production profile, focusing on interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) of human CD4+, CD8+ and CD4- CD8- alpha beta T cells cloned from the peripheral blood of healthy individuals was compared. Solid-phase anti-CD3 stimulation of CD4- CD8- alpha beta T cell clones from one individual revealed a significantly increased frequency of IL-4-producing clones (81%), compared to CD4+ T cells (24%) or CD8+ (28%). All five CD4- CD8- alpha beta T-cell clones from two other individuals also produced IL-4. Clones that produced IFN-gamma with undetectable IL-4 production, thus being of the 'classical' Th1 phenotype, were infrequent in CD4- CD8- alpha beta T-cell clones (19%) compared to CD4+ (71%), and CD8+ clones (72%) cloned in OKT3, and CD4+ cells cloned in phytohaemaglutinin A (77%). Unlike previously reported studies with gamma delta cells, the alpha beta CD4- CD8- T cells produced IL-10 at appreciable frequency (38%) in PHA generated clones. The supernatants from anti-CD3 stimulated CD4- CD8- alpha beta T-cell clones contained sufficient IL-4 to activate B cells, enhancing CD23 and surface immunoglobulin M (IgM) expression and co-stimulating B-cell proliferation. These findings suggest that the function of CD4- CD8- alpha beta T cells is distinct from that of most CD4+ or CD8+ T cells.

IL-10 enhances expression of the IL-2 receptor alpha chain on T cells.

Interleukin-10 (IL-10) has various immunomodulatory actions depending on the target cell type. Some of these effects have been shown to be owing to its ability to down-regulate surface expression of markers, for example HLA-DR on macrophages and CD25 (IL-2 receptor alpha chain) on B cells. In this report we show that preincubation of IL-10 for 24 hr up-regulates expression of the activation marker CD25, but not HLA-DR on cloned T cells of various phenotypes such as CD4+, CD8+, CD4- CD8- alpha beta and gamma delta T-cell receptor (TCR)-expressing cells. This up-regulation of CD25 was accompanied by an increase in the T cells IL-2-dependent proliferative response in 63% of the CD4+ clones and 100% of the CD8+, CD4-, CD8- alpha beta and gamma delta TCR+ clones analysed. IL-10 was also shown to be at least partly responsible for the up-regulation of CD25 on mitogen-activated peripheral blood mononuclear cells, suggesting that IL-10 has this CD25 modulatory effect within a more physiological environment. Our data suggest that IL-10 can have a multitude of effects on human T cells, and should not be considered exclusively as an immunoinhibitory cytokine.

Immunoregulatory role of interleukin 10 in rheumatoid arthritis.

The presence and the role of interleukin 10 (IL-10), a potent cytokine synthesis inhibitory factor and antiinflammatory cytokine, were investigated in rheumatoid arthritis (RA). The expression of both mRNA and protein for IL-10 could be demonstrated in RA and osteoarthritis (OA) joints. Human IL-10 mRNA could be demonstrated by polymerase chain reaction amplification of cDNA made by reverse transcription of total RNA extracted directly from synovial tissue in five out of five RA and four out of five OA patients. IL-10 protein was demonstrated by specific immunoassay and immunohistology. IL-10 protein was spontaneously produced in all 11 RA and 17 OA synovial membrane cultures investigated, and this production was sustained for up to 5 d in culture in the absence of any extrinsic stimulation. IL-10 protein could also be detected by immunohistology in all five RA and four OA synovial membrane biopsies investigated, but not three normal synovial membranes. Immunohistology revealed that the IL-10 was localized to the synovial membrane lining layer and mononuclear cell aggregates. Immunofluorescence double staining revealed that the sources of IL-10 were monocytes in the lining layer, and T cells in the mononuclear cell aggregates. We found evidence that the IL-10 expression was functionally relevant, as neutralization of endogenously produced IL-10 in the RA synovial membrane cultures resulted in a two- to threefold increase in the protein levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and IL-1 beta, although IL-6 and IL-8 levels were not affected. The addition of exogenous recombinant IL-10 to the RA synovial membrane cultures resulted in a two- to threefold decrease in the levels of TNF-alpha and IL-1 beta. IL-8 levels were reduced by day 5; however, IL-6 levels were not affected by exogenous IL-10. Neutralization of the endogenous IL-10 in two out of seven RA synovial membrane cultures resulted in the expression of detectable levels of interferon gamma (561-1,050 pg/ml). Taken together, the above findings suggest that IL-10 is spontaneously produced in RA and OA and is an important immunoregulatory component in the cytokine network of RA, regulating monocyte and in some cases T cell cytokine production.

Evaluation of the role of cytokines in autoimmune disease: the importance of TNF alpha in rheumatoid arthritis.

Cytokines and growth factors are involved in all important biological processes. Hence it is anticipated that they will be of importance in autoimmune disease. The pathogenesis of autoimmune diseases involves a number of stages, initiation, perpetuation and tissue damage, each of which involves different cell and molecular interactions. In this review, we will discuss an outline of the cytokine involvement in the various stages of autoimmune development, prior to focusing on the analysis of cytokines in rheumatoid arthritis. Cytokines exert their effect via high affinity cell surface receptors. Thus an understanding of cytokines involves the analysis of receptor expression, and also of cytokine inhibitors. Currently there is only adequate knowledge of these aspects in rheumatoid arthritis (RA), and as such the emphasis of this review is on RA. One of the major reasons for being interested in the role of cytokines in autoimmunity is to define possible therapeutic targets. There is now considerable evidence that TNF alpha is such a target in RA, and the effect of anti TNF alpha monoclonal antibody therapy in RA is discussed.