Effects of interferon-gamma and tumour necrosis factor-alpha on the development of cytotoxic T lymphocytes in autologous mixed lymphocyte tumour cultures with human melanoma.

We have studied the influence of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the development of cytotoxic T lymphocytes (CTL) against melanoma in mixed lymphocyte tumour cultures (MLTC). In these MLTC, TNF-alpha at 10(4) U/ml increased the expansion of the CTL up to 10(4)-fold over recombinant IL-2 (rIL-2) alone. IFN-gamma at 10(4) U/ml and combinations of TNF-alpha plus IFN-gamma at 10(2)-10(3) U/ml promoted the proliferation more variably. MLTC generated with rIL-2 showed a predominance of CD8+ cells, while 2 weeks of culture in the presence of IFN-gamma at 10(4) U/ml, or with IFN-gamma and TNF alpha at 1 x 10(2)-10(3) U/ml, favoured the emergence of CD4+ cell populations. The cytotoxic activity of the lymphocytes generated in these MLTC showed a consistent decline of K562 cytotoxic activity following exposure to the combination of IFN-gamma and TNF-alpha. Despite the altered T cell subset distribution with different combinations of cytokines, no consistent alteration in the specific anti-tumour cytotoxicity against melanoma was detected. These results suggest that TNF-alpha and IFN-gamma influence the activation, phenotypic, and functional outcome of MLTC-generated CTL, and may account for the phenotypic variations observed in T cell populations generated in vitro.     

A novel strategy for targeting CD4+ PPD-reactive T cells against tumour cells using PPD monoclonal antibody heteroconjugates.

We have constructed PPD monoclonal antibody heteroconjugates specific for a tumour-associated antigen of C57BL/6 melanomas or for human complement component C3d fixed de novo to murine fibrosarcoma cells (MC6A). The ability of our heteroconjugates to target CD4+ PPD-reactive T cells against the appropriate tumour targets was then determined in vitro. Heteroconjugate-treated B16-F10 and MC6A tumour targets were both able to present PPD to the specific T cells, resulting in activation and concomitant lymphokine secretion. Secreted lymphokines were then demonstrated to cause significant tumour cytolysis and cytostasis in vitro. Preliminary experiments in vivo suggest that this targeting system may provide the basis for a future immunotherapeutic strategy.     

Effect of interferon-gamma priming on the activation of murine peritoneal macrophages to tumouricidal state by cisplatin, IL-1, and tumour necrosis factor (TNF): production of IL-1 and TNF.

The effect of interferon-gamma (IFN-gamma) priming of murine peritoneal macrophages on the activation to tumouricidal state by cisplatin, lipopolysaccharide (LPS)-IL-1 and TNF was investigated. Cisplatin-, LPS-, IL-1- or TNF-treated IFN-gamma-primed macrophages showed significantly enhanced tumouricidal activity and binding to tumour cells, compared with unprimed treated or untreated macrophages. Macrophages treated with cisplatin, LPS, IL-1 and TNF produced released and membrane-associated IL-1 and TNF activity which was significantly enhanced after priming with IFN-gamma. These observations suggest the use of IFN-gamma along with these biological response modifiers in designing immunotherapeutic protocols for treatment of malignancy.     

Effect of lipoarabinomannan and mycobacteria on tumour necrosis factor production by different populations of murine macrophages.

Tumour necrosis factor (TNF) production is an important pathological mediator in mycobacterial infections, and yet little is known of the factors which influence its production. We have studied the influence of murine macrophage heterogeneity and activation state on TNF production following mycobacterial stimulation in vitro. Lipoarabinomannan (LAM) from strains of Mycobacterium tuberculosis and Myco. avium differentially stimulated TNF production in thioglycollate-elicited macrophages in a dose-dependent manner. In comparison, resident peritoneal macrophages produced much less TNF when stimulated with LAM, dead mycobacteria or lipopolysaccharide (LPS). In contrast, zymosan stimulated resident macrophages to a higher degree than thioglycollate-elicited cells. Another comparison between bone marrow and thioglycollate-elicited macrophages showed that both responded to LPS, but only the latter was stimulated significantly by H37Rv LAM. This may indicate that LAM stimulation of macrophages takes place through a different pathway than both zymosan- and LPS-stimulated TNF production. Also, in vitro activation of peritoneal macrophages with interferon-gamma (IFN-gamma), increased TNF response to several stimuli. Our studies indicate that the pathology of mycobacterial infections through TNF production may be influenced by the type and activation state of the macrophage which responds to that infection.     

Augmentation of the human monocyte/macrophage chemiluminescence response during short-term exposure to interferon-gamma and tumour necrosis factor-alpha.

The effects of short-term (30 min) pre-incubation of human monocytes and macrophages (3-day cultured monocytes) with leucocyte-derived human interferon-gamma (IFN-gamma) and recombinant human tumour necrosis factor-alpha (rTNF-alpha) were examined. Pre-incubation of either monocytes or macrophages with rTNF-alpha or IFN-gamma (100 U/5 x 10(5) cells) augmented their respiratory burst to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), measured by the luminol- and lucigenin-dependent chemiluminescence assay. In addition, both cell types showed a burst of respiratory activity in the presence of rTNF-alpha or IFN-gamma only. The effects of IFN-gamma were removed by adsorption with an anti-IFN-gamma monoclonal antibody and those of rTNF-alpha were abolished by heating at 100 degrees C, or by the addition of anti-TNF-alpha monoclonal antibody. The results demonstrate that both IFN-gamma and rTNF-alpha are stimulators of monocytes and macrophages, and rapidly alter the capacity of the cells to respond to fMLP, which binds to cell surface receptors.     

Triggered human mucosal T cells release tumour necrosis factor-alpha and interferon-gamma which kill human colonic epithelial cells.

T cell activation can lead to local tissue injury in organ culture studies of human fetal jejunum, either directly through cytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cytotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions are required for killing of human colonic epithelial cells. Cytokine-containing supernatants were induced by incubating unseparated lamina propria lymphocytes (LPL) or mucosal T cell subpopulations (separated by indirect panning) with anti-CD3 and/or K562 target cells for 18 h at 37 degrees C. Cytokines were measured by cytotoxicity assays using L929 (murine fibroblast) and HT-29 (human colonic tumour) lines as target cells in combination with blocking anti-cytokine antibodies. Supernatants derived from unseparated, CD4+ (greater than 95% pure) and CD8+ (greater than 90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/ml tumour necrosis factor-alpha, respectively). All or nearly all of the cytotoxicity was due to the presence of tumour necrosis factor-alpha (little or no tumour necrosis factor-beta was detected). These same supernatants were cytotoxic (up to 32% lysis at 1/4 dilution) to HT-29 targets in a 48-h 111In release assay. Recombinant tumour necrosis factor-alpha and interferon-gamma alone produced minimal killing of HT-29, but together killed the HT-29 target cells. Anti-tumour necrosis factor-alpha or anti-interferon-gamma alone blocked killing of HT-29 target cells by LPL-derived supernatants, although anti-tumour necrosis factor-beta had no effect upon killing of HT-29. These results demonstrate that human LPL T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosal damage in chronic inflammatory states such as inflammatory bowel disease.     

Progranulin promotes tumour necrosis factor‐induced proliferation of suppressive mouse CD4+ Foxp3+ regulatory T cells

Progranulin (PGRN) is a pleiotropic growth factor with immunosuppressive properties. Recently, it was reported that PGRN was an antagonist of tumour necrosis factor (TNF) receptors, preferentially for TNFR2. However, we and others showed that TNF–TNFR2 interaction was critical for the activation and expansion of functional CD4⁺ Foxp3⁺ regulatory T (Treg) cells. We therefore examined the effect of PGRN on the proliferation of naturally occurring murine suppressive Treg cells induced by TNF. Consistent with our previous reports, TNF overcame the hyporesponsiveness of highly purified Treg cells to T‐cell receptor stimulation. Furthermore, in the presence of interleukin‐2, TNF preferentially stimulated proliferation of Treg cells contained in unfractionated CD4 cells. These effects of TNF on suppressive Treg cells were markedly increased by exogenous PGRN. TNF and TNFR2 interactions are required for this effect of PGRN, because the PGRN by itself did not stimulate Treg cell proliferation. The effect of PGRN on Treg cells was abrogated by antibody against TNFR2, and Treg cells deficient in TNFR2 also failed to respond to PGRN. Furthermore, PGRN also enhanced the proliferative responses of effector T cells to TNF, but to a lesser extent than that of Treg cells, presumably caused by the different levels of TNFR2 expression on these two subsets of CD4 cells. Hence, our data clearly show that PGRN promotes, rather than inhibits, the functional consequence of TNF–TNFR2 interaction on Treg cells.     

Higher serum levels of tumour necrosis factor and its soluble receptors are associated with ovarian tumours

Introduction

Tumour necrosis factor (TNF) and its soluble receptors type 1 (sTNF-R1) and type 2 (sTNF-R2) have been suggested as key mediators between apoptosis and cancer cell progression. The aim was to examine concentrations of the parameters in the serum of women with ovarian tumour and in the fluid from ovarian cysts of women with serous cystadenoma.

Material and methods

The study included 125 women with ovarian tumours. As a control, sera were obtained from 70 healthy female volunteers. Concentrations of TNF, sTNF-R1 and sTNF-R2 were measured by enzyme-linked immunosorbent assay (ELISA).

Results

Significant increases of TNF, sTNF-R1 and sTNF-R2 were found in the serum of women with ovarian tumour in comparison to the control (p < 0.0001). The highest levels of all studied parameters were observed in women with ovarian cancer. In the ovarian cyst fluid the concentrations of the evaluated parameters increased significantly as compared to the serum (p < 0.0001).

Conclusions

Our data showed changes in regulatory mechanisms of apoptosis in women with ovarian tumours which are associated with increased concentrations of all studied factors. Serum estimated TNF and especially sTNF-R may be used as complementary diagnostic markers in patients with ovarian tumours.     

Altered production of tumour necrosis factors alpha and beta and interferon gamma by HIV-infected individuals.

In vitro studies shows that recombinant tumour necrosis factor (TNF) alpha and beta, and interferon-gamma (IFN-gamma) can enhance HIV replication, and peripheral blood mononuclear cells (PBMC) infected with HIV in vitro secrete high levels of the same cytokines. As T cells secrete all three mediators, the capacity of T cell activation signals to trigger cytokine production in PBMC from HIV-infected individuals was investigated as such patients may be immunocompromised. We demonstrate that asymptomatic seropositives in CDC group II/III as well as patients who have progressed to CDC group IV of the disease proliferate efficiently to anti-CD3 antibody, recombinant interleukin-2 (rIL-2), phytohaemagglutinin (PHA), PHA plus phorbol 12,13 dibutyrate (PMA) but secrete significantly (P less than 0.05) higher amounts of TNF-alpha, TNF-beta and IFN-gamma compared with controls in response to the same stimulants. We also show a difference between group II/III and group IV patients with the latter secreting more TNF-alpha and IFN-gamma. The kinetics of TNF-alpha and -beta, and IFN-gamma production was stimulus dependent with overall levels varying in time for each stimulus. Furthermore, the kinetics of the response to all three stimulants were altered in seropositives; CDC group II/III and group IV patients secreted higher levels of cytokines over several time points compared to controls. The altered production of these mediators by HIV-infected patients may contribute to disease progression and to the pathogenesis of AIDS.     

Soluble tumour necrosis factor receptors (sTNF-R) and HIV infection: correlation to CD8+ lymphocytes.

The objective of this study was to determine sTNF-R, type I (p55) and type II (p75) in sera of HIV-infected male homosexuals and correlate them to T lymphocyte subpopulations and course of HIV infection. Serum samples were obtained from 39 HIV-1+ asymptomatic male homosexuals, 10 symptomatic (ARC and AIDS) male homosexuals and 44 HIV- non-homosexual healthy controls. sTNF-R levels were determined by ELISA with specific MoAbs and polyclonal antibodies to the sTNF-R proteins. sTNF-RI and II levels were significantly elevated in 72% and 74% respectively of HIV+ asymptomatic male homosexuals and in all of the symptomatic male homosexuals. In sequential studies a highly significant positive correlation was found between sTNF-RI and sTNF-RII (r = 0.8, P < 0.001) and between both sTNF-R and CD8+ lymphocyte counts (r = 0.6 and 0.92, respectively, P < 0.01-0.001) during the asymptomatic stage of the infection. All these correlations were lost, however, during the symptomatic phase of the disease. These results suggest that: (i) HIV infection is associated with elevation of sTNF-R serum levels; (ii) sTNF-R levels are strongly correlated to CD8+ lymphocytes during the asymptomatic stage of HIV infection.     

Tumour necrosis factor-alpha and interferon-gamma production measured at the single cell level in normal and inflamed human intestine.

The spot-ELISA technique has been used to enumerate the frequency of cells secreting tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), isolated from biopsies of normal intestine and from biopsies of children with inflammatory bowel disease. TNF-alpha production was undetectable in six out of 12 biopsies from normal intestine and in the other six biopsies it ranged from 60 to 580 TNF-alpha-secreting cells/10(6) isolated intestinal cells. In contrast, cells isolated from biopsies of children with Crohn's disease (n = 9) all showed elevated frequencies of TNF-alpha-secreting cells (500-12,000 secreting cells/10(6) cells). In ulcerative colitis, four out of eight children had increased production of TNF-alpha and in children with indeterminate colitis two out of three had elevated levels. There was no correlation between plasma TNF-alpha levels and the number of intestinal cells secreting TNF-alpha. In controls and all groups of patients IFN-gamma-secreting cells were uncommon. These results suggest that TNF-alpha is an important mediator of inflammation in the human gut, and, furthermore, may play a role in the growth failure frequently seen in children with inflammatory bowel disease.     

Tumour necrosis factor (TNF) production by T cell receptor-primed T lymphocytes is a target for low dose methotrexate in rheumatoid arthritis.

Methotrexate (MTX) is an effective immunosuppressive agent in various chronic inflammatory diseases such as rheumatoid arthritis (RA). However, its mechanisms of action are only partially understood. In this study, we assessed the effects of MTX on the differentiation of peripheral blood (PB) CD4+CD45RA 'naive' and CD4+CD45RO 'memory' T cells from healthy controls and patients with RA. Accordingly, purified T cells were primed and restimulated in vitro via the T cell receptor (TCR) in the presence of IL-2 to generate effector T cells secreting large amounts of Th1 and Th2 cytokines. We observed that low doses of MTX strongly suppress TNF and to a lesser extent interferon-gamma (IFN-gamma) production by T cells from both healthy donors and RA patients when present during T cell priming via the TCR. Similar data were obtained for TCR-primed synovial fluid mononuclear cells in RA. In contrast, production of IL-4 by TCR-primed CD45RA T cells was significantly increased upon MTX treatment. Interestingly, MTX did not enhance IL-4 production when present during restimulation of effector CD45RO T cells, although it still suppressed TNF production. The results indicate that MTX effects depend on the stage of T cell activation and identify TNF production by TCR-primed T lymphocytes as a target for low-dose MTX treatment in RA. These findings could explain the delayed clinical effects of MTX and may contribute to its potent anti-inflammatory and immunoregulatory properties.     

A model for the investigation of factors influencing haemorrhagic necrosis mediated by tumour necrosis factor in tissue sites primed with mycobacterial antigen preparations.

Mycobacterial lesions and skin sites challenged with soluble mycobacterial antigen are very sensitive to the necrotizing effect of tumour necrosis factor (TNF). We have used a model that permits separate quantitative assessment of swelling and haemorrhage to show that when these reactions are elicited in mice that have not been deliberately immunized, pretreatment of the mice with lipopolysaccharide (LPS), or with a MoAb to CR3 which blocks emigration of myeloid cells into the tissues, will block both the swelling and the haemorrhage. On the other hand, treatment with an inhibitor of platelet-activating factor (PAF), or with misoprostol (a synthetic prostaglandin E1 analogue), or with cobra venom factor (CVF) which depletes complement, preferentially blocks the haemorrhagic component, while leaving the swelling relatively unaltered. As swelling occurs before the haemorrhage is seen, it is possible that these factors act at a late stage in the cascade of events leading to the tissue damage. However, LPS and CVF were able to inhibit swelling and haemorrhage in the massive reactions elicited in pre-immunized animals, whereas the PAF inhibitor had no detectable effect.     

Human polymorphonuclear leucocytes stimulated by tumour necrosis factor-alpha show increased adherence to extracellular matrix proteins which is mediated via the CD11b/18 complex.

The present study demonstrates that tumour necrosis factor (TNF) and FMLP, but not IL-1 or IL-8, enhanced the adherence of polymorphonuclear neutrophil (PMN) to fibronectin, an extracellular matrix protein. The adherence induced by FMLP was very rapid, within 5 min while the induction of adherence by TNF was much slower, reaching maximum at 60 min. TNF also enhanced an adhesion of PMN to other extracellular matrix proteins, such as laminin, collagen IV and gelatin II, but not to human serum albumin. Anti-CD18 MoAb completely inhibited the binding of TNF-stimulated PMN to fibronectin and partially inhibited the binding to laminin. Further investigation showed that adhesion of TNF-stimulated PMN to fibronectin and laminin was inhibited by anti-CD11b MoAb and to a lesser extent by CD11a MoAb. In contrast to TNF-stimulated PMN the binding of unstimulated PMN to fibronectin and laminin was only inhibited by anti-CD11a MoAb. Anti-CD11c had no effect on PMN adherence. These results suggest that unstimulated PMN adhere to extracellular proteins through the CD11a/18, while TNF-stimulated PMN adhere through the CD11b/18. These results suggest that TNF secreted at the site of inflammation may enhance the interaction of PMN with the extravascular environment through the CD11b/18 complex.     

The role of tumour necrosis factor-alpha and IL-1 in polymorphonuclear leucocyte and T lymphocyte recruitment to joint inflammation in adjuvant arthritis.

The mediators involved in leucocyte recruitment to joints during arthritis are not fully defined, but two important proinflammatory cytokines, IL-1 and tumour necrosis factor-alpha (TNF-alpha), are produced in joints in rheumatoid arthritis (RA). We investigated in the rat adjuvant arthritis model whether endogenous IL-1 and TNF-alpha contribute to joint inflammation and polymorphonuclear leucocyte (PMNL) and T lymphocyte infiltration. The migration of 51Cr-labelled rat blood PMNL and 111In-labelled T lymphocytes to the joints of rats with adjuvant arthritis was measured along with plasma protein extravasation, which was quantified using 125I-labelled human albumin. Rats with active arthritis of 5 days' duration received i.p. non-immune serum, polyclonal neutralizing anti-serum to rat TNF-alpha, antiserum to IL-1 alpha and IL-1 beta, or both anti-TNF plus anti-IL-1 for 5 days. Treatment with anti-IL-1 alpha and IL-1 beta did not affect plasma protein extravasation, or PMNL or T lymphocyte accumulation in the joints (i.e. talar joint, hind paws, and tail) despite the fact that this treatment inhibited 80-90% of the PMNL migration into dermal sites injected with IL-1 alpha or IL-1 beta. In contrast, anti-TNF-alpha treatment significantly improved clinical scores, decreased plasma protein extravasation by 60-80%, inhibited PMNL accumulation by 40-50% and decreased T lymphocyte accumulation by 30-50%. Treatment with anti-IL-1, together with anti-TNF-alpha, significantly potentiated the inhibition of T lymphocyte accumulation observed with anti-TNF-alpha alone. These results indicate that endogenous TNF-alpha production may play an important role in the inflammatory changes and leucocyte recruitment in this experimental model of human arthritis, while IL-1 may have a less important role in leucocyte recruitment to these joints.     

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) induced in human CD4⁺ and CD8⁺ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4⁺ cells can make IL-2 than the other two cytokines, and that there are more TNF-α-positive CD4⁺ cells than cells with IFN-γ. For normal CD8⁺ cells the highest production of cytokine is of IFN-γ (up to 50% of the cells) especially at longer times (10–20h) of stimulation. For CD8⁺ cells, IL-2-positive cells exceed those with TNF-α. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4⁺ or CD8⁺ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-α but significantly raised levels of production of IFN-γ in both CD4⁺ and CD8⁺ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.     

Growth and major histocompatibility antigen expression regulation by IL-4, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) on human renal cell carcinoma

We have recently shown that human renal cell carcinoma (RCC) tumour lines express high-affinity IL-4 receptors. Binding of IL-4 to RCC cells induced a growth inhibition in the range of 20 68%. To enhance the growth inhibitory effect of IL-4. we have tested the effects of two additional cytokines capable of directly affecting tumour cell growth. IFN-γ caused a significant inhibition of RCC tumour cell growth (up to 70%) in a dose-dependent manner, whereas the effect of TNF-α was more limited (0 20% inhibition). The addition of IL-4 to IFN-γ on RCC cells sensitive to lL-4 induced a greater inhibition of cell growth than that seen with each cytokine alone. IL-4 and IFN-γ rendered RCC cells more responsive to the inhibitory effect mediated by TNF-α, The combination of TNF-Q with IL-4 and IFN-γ induced an optimal growth inhibition (up lo 90 98%) of RCC cells. In addition to a direct anti-proliferative effect, we have demonstrated that these cytokines can also enhance the expression of MHC antigens on the surface of RCC tumour cell lines which may render the cells more immunogenic, All RCC lines tested expressed class 1 antigens, but not class II antigens. IFN-γ induced class II expression and up-regulated the expression of class I antigens on RCC cells. Class II antigen expression was detectable following 48 h incubation, and greater after 72 h with IFN-7. lL-4 minimally affected class I expression, whereas TNF-(v up-regulated class I antigen expression. IL-4 or TNF-α did not induce class II expression. The combination of The three cytokines slightly augmented the up-regulation of class I and class II antigens observed with IFN-γ alone. These observations confirm the direct interaction of IL-4, IFN-γ and TNF-a with RCC tumour cells. both at the level of growth regulation and MHC antigen expression, and suggest a therapeutic potential of the combination of the three cytokines for renal ceil carcinoma.     

Characterization of CD4+ T helper cells in patients with Kawasaki disease (KD): preferential production of tumour necrosis factor-alpha (TNF-alpha) by V beta 2- or V beta 8- CD4+ T helper cells.

KD is an acute febrile illness in children characterized by coronary arteritis accompanied by aneurysm and thrombotic occlusion. The etiology of KD is unknown. It has been recently reported that KD is associated with the selective expansion of V beta 2+ and V beta 8.1+ T cells in peripheral blood lymphocytes (PBL), by studying the T cell receptor (TCR) repertoire of in vitro activated T cells. KD may therefore be caused by a superantigen [1-3]. To understand better the immunopathology of KD, we investigated TCR V beta 2 and V beta 8.1 expression on both the T cells of freshly isolated PBL and T cell clones (TCC) from patients with KD. Cytokine production by TCC was also studied. Blood samples were obtained from patients with acute (n = 20) and convalescent (n = 20) KD, age-matched children with non-infectious diseases (n = 18), and healthy adults (n = 20). Among these four groups, there were no significant differences in the percentages of either V beta 2+ or V beta 8.1+ T cells of freshly isolated PBL. The same was true for the CD4+ or CD8+ T cell subsets. One hundred and five TCC (98 CD3+ CD4+ CD8- and seven CD3+ CD4- CD8+) established from the affected skin, lymph node or PBL of six patients with KD were also negative for either V beta 2 or V beta 8.1 TCR. Sixty-eight of 105 TCC (65%) produced detectable levels (> 5 pg/ml) of TNF-alpha (6-1016 pg/ml), in the absence of any stimuli. In contrast, only 11 (10%) of 105 TCC or 7 (7%) of 97 TCC produced detectable levels of IL-2 or IL-6, respectively, in the absence of any stimuli. Stimulation with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) induced most TCC to produce higher amounts of TNF-alpha, IL-2 and IL-6. These results suggest that CD4+ T helper cells expressing TCR-beta other than V beta 2 or V beta 8 receptor, primarily through TNF-alpha production, are involved in the immunopathology of KD.     

Methylprednisolone differentially regulates IL-10 and tumour necrosis factor (TNF) production during murine endotoxaemia

IL-10 is an endogenous antiinflammatory cytokine that inhibits TNF biosynthesis and protects mice from lipopolysaccharide (LPS)-induced lethality. As synthetic glucocorticoids are widely used as antiinflammatory agents, we analysed the effects of methylprednisolone administration on IL-10 biosynthesis during murine endotoxaemia. We found that low doses of methylprednisolone (2–10 mg/kg) markedly inhibited TNF production but did not affect serum levels of IL-10, while a high methylprednisolone dose (50 mg/kg) increased LPS-induced IL-10 levels. In parallel, we observed that LPS-induced IL-10 production is TNF-independent in this experimental setting. Experiments conducted in vitro indicated that methylprednisolone (from 0·01 to 100 μg/ml) also increased the biosynthesis of IL-10 by LPS-activated mouse peritoneal macrophages. We conclude that methylprednisolone differentially regulates IL-10 and TNF production induced by LPS both in vivo and in vitro at the macrophage level.     

Elevated serum levels of soluble tumour necrosis factor receptors (sTNF-R) in patients with HIV infection.

Serum levels of the soluble form of tumour necrosis factor receptor type II (p75) (sTNF-R) were determined in HIV-infected individuals and risk groups and were then correlated with the course of infection and prognosis. sTNF-R levels were determined by an ELISA with MoAbs and polyclonal antibodies to urine-derived sTNF-R proteins. The mean +/- s.e. levels of sTNF-R in the sera of 49 HIV+ male homosexuals, 34 HIV- male homosexuals and 44 matched controls were 6.1 +/- 0.3 ng/ml, 4.4 +/- 0.3 ng/ml and 3.4 +/- 0.2 ng/ml, respectively. All these values were significantly different between each of the groups (P less than 0.001-0.05). Sequential studies of sTNF-R revealed higher levels following seroconversion in 5/8 individuals, remained persistently high during the asymptomatic phase of the infection and became even more elevated in some ARC and AIDS patients. At the same time TNF-alpha was undetectable in sera obtained from HIV+ male homosexuals and from healthy controls. This was independent of stage of HIV infection, serum sTNF-R level and type of ELISA kit used. These findings suggest that TNF-alpha/TNF-R system is turned on before and during HIV infection and raise the possibility that sTNF-R, the natural inhibitor of TNF, may be of importance in determining the course and probably prognosis of the disease.