Interferon-gamma, tumor necrosis factor-alpha, and interleukin 1-beta suppress the replication of hepatitis B virus through oxidative stress

The effect of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and H2O2, on hepatitis B virus (HBV) replication was analyzed in the HBV DNA transfected human hepatoblastoma-derived cell line HB 611. Secretion of HBV DNA from HB611 cells was inhibited by IFN-gamma, TNF-alpha, IL-1beta, and H2O2 in a dose-dependent manner. These cytokines and H2O2 also decreased HBV mRNA expression in HB611 cells, meaning that these reagents decreased the synthesis of all virally encoded components of the HBV virion. The level of manganese-SOD mRNA, indicative of occurrence of oxidative stress, increased immediately after treatment with IFN-gamma, TNF-alpha, IL-1beta, and H2O2. Moreover, the antioxidant N-acetyl-L-cysteine substantially inhibited the antiviral effect. Our findings suggest that oxidative stress may be a common factor in the antiviral effects of IFN-gamma, TNF-alpha, and IL-1beta on HBV.     

Renal allograft rejection: induction and function of adhesion molecules on cultured epithelial cells.

The interaction of graft-infiltrating immune cells with donor parenchymal cells is an important early event in allograft rejection. This binding is stabilized by interaction of antigen-independent 'adhesion' molecules expressed on the two cell types. As the level of expression of these molecules can be altered during inflammation, a series of experiments was performed to examine the effects of the inflammatory cytokines interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on adhesion molecules expressed by cultured human renal tubular epithelial cells. These cells constitutively expressed ICAM-1 and LFA-3. Incubation with IFN-gamma increased expression of ICAM-1 but had no significant effect on expression of LFA-3 (P greater than 0.05). Incubation with TNF-alpha increased expression of both ICAM-1 and LFA-3; IFN-gamma synergized with TNF-alpha to further augment expression of these molecules. Peripheral blood lymphocytes (PBL) showed an enhanced binding to allogeneic renal epithelial cell monolayers which had been pretreated with IFN-gamma or TNF-alpha. MoAbs specific for ICAM-1 or its ligand LFA-1 inhibited adhesion of PBL to either IFN-gamma- or TNF-alpha-pretreated renal cells. By contrast, antibodies specific for LFA-3 or its ligand CD2 only significantly blocked PBL adhesion to renal cells which had been pretreated with TNF-alpha. Combination of antibodies specific for multiple components of the adhesion systems produced greater inhibition of adhesion than was produced by any single MoAb. These results suggest that the inflammatory cytokines IFN-gamma and TNF-alpha up-regulate expression of functional ICAM-1 and LFA-3 molecules which can augment the binding of potentially graft-damaging lymphoid cells to renal tubular epithelial cells.     

Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture

In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1 (IL-1 alpha and beta) and interleukin-2 (IL-2) in the presence or absence of thyroid-stimulating hormone (TSH). Neither TNF-alpha nor IL-2 affected thyroid cell survival, IFN-gamma was usually inhibitory and IL-1 alpha slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN-gamma, IL-2 and TNF-alpha inhibited the ability of thyroid lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well-recognized ability to induce MHC class II antigens on thyroid cells, IFN-gamma may inhibit thyroid cell proliferation and TNF-alpha, IFN-gamma and IL-2 may down-regulate thyroid autoantibody synthesis.     

Production of interleukin (IL)-5 and IL-10 accompanies T helper cell type 1 (Th1) cytokine responses to a major thyroid self-antigen, thyroglobulin, in health and autoimmune thyroid disease

Tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma exert detrimental effects in organ-specific autoimmune disease, while both destructive and protective roles have been demonstrated for interleukin (IL)-10, IL-4 and IL-5. We examined the production of these cytokines by peripheral blood mononuclear cells (PBMC) from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and healthy controls, upon exposure to a thyroid self-antigen, human thyroglobulin (Tg), in the presence of autologous serum. Initially, TNF-alpha and IL-2 were produced in all three groups, accompanied by IL-10. Release of IFN-gamma, IL-4 and, notably, IL-5 ensued. Both patient groups exhibited increased TNF-alpha, IL-2, IFN-gamma and IL-10 responses, and PBMC from HT patients secreted lower amounts of IL-5 than male, but not female, controls. Enhanced TNF-alpha production by HT cells also occurred in the presence of pooled normal sera, indicating a dependency on intrinsic cellular factors. Conversely, higher production of TNF-alpha and IL-5 occurred in the presence of autologous sera than in the presence of pooled normal sera in both patient groups, indicating a dependency on serum constituents. Complement appeared to promote the production of IL-2 and particularly IL-5, the levels of which were reduced by neutralization of complement by heat- or zymosan treatment. The production of IFN-gamma and IL-2 of the three groups together correlated directly with the serum anti-Tg activity. Moreover, TNF-alpha, IFN-gamma, IL-5 and IL-10 responses were markedly inhibited by partial denaturation of Tg by boiling. We hypothesize that autoantibodies and complement may promote mixed Th1/Th2 cell cytokine responses by enhancing the uptake of autoantigens by antigen-presenting cells.     

Induction of thymocyte apoptosis by systemic administration of concanavalin A in mice: role of TNF-alpha, IFN-gamma and glucocorticoids

Administration of concanavalin A (Con A) is a well-established model of acute immune-mediated hepatitis. Here, we demonstrate that intravenous injection of Con A in mice induces profound thymic atrophy. Compared to liver damage, the kinetics of Con A-induced thymic atrophy is slower and more prolonged; the nadir in thymocyte number is reached 4 days after Con A injection, whereas peak transaminase levels are observed at 12-24 h. Marked alterations in the ratio of CD4+ and CD8+cells in the thymus and spleen and significantly increased rates of thymocyte and splenocyte apoptosis are observed. Neutralization of the cytokines TNF-alpha or IFN-gamma, which protects mice from Con A-induced hepatitis, prevents thymic atrophy as well as alterations in CD4+ and CD8+ cell numbers and apoptosis rates. However, neither TNF-alpha nor IFN-gamma are detectable in thymocyte lysates after Con A injection, whereas both cytokines are present in liver, spleen and serum. Administration of the glucocorticoid receptor antagonist mifepristone does not prevent thymic atrophy, thus ruling out a possible contribution of endogenous glucocorticoids. Con A-induced thymic atrophy is accompanied by down-regulation of Bcl-2 expression in the thymus, which is prevented by neutralization of TNF-alpha or IFN-gamma. These data demonstrate that the thymus is a critical target organ of Con A-induced inflammation; the effects of Con A on the thymus are mediated by extrathymic production of TNF-alpha and IFN-gamma, but not by glucocorticoids.     

Ebastine inhibits T cell migration, production of Th2-type cytokines and proinflammatory cytokines

BACKGROUND: Cytokine imbalance and cellular migration to inflammatory sites are critical components of allergic diseases. Redirecting cytokine imbalance and inhibiting cell migration therefore represent important therapeutic strategies for the treatment of these disorders. OBJECTIVES: To study the in vitro effect of ebastine, a novel non-sedating H1 receptor antagonist, on cytokine secretion and migration of activated T cells, as well as production of pro-inflammatory cytokines by macrophages. METHODS: Peripheral T cells obtained from healthy volunteers were cultured in wells coated with the combination of anti-CD3 monoclonal antibody (mAb) and anti-CD26 mAb, anti-CD3 mAb and anti-CD28 mAb, or anti-CD3 mAb with PMA, in the presence or absence of ebastine. T cell proliferation and the production of cytokines were measured by [3H]thymidine incorporation assay and ELISA, respectively. In addition, transendothelial migration of T cells and production of pro-inflammatory cytokines by macrophages were examined. RESULTS: Ebastine inhibited T cell proliferation and the production of IL-4, IL-5, IL-6, and TNF-alpha by T cells under each co-stimulatory condition tested, whereas it exhibited no effect on the production of IL-2 or IFN-gamma. In addition, T cell migration and the production of such pro-inflammatory cytokines as TNF-alpha and IL-6 by macrophages were inhibited by ebastine. CONCLUSIONS: These results indicate that ebastine has a specific inhibitory effect on Th2-type cytokine production. Moreover, ebastine inhibited T cell migration and pro-inflammatory cytokine production by T cells and macrophages, suggesting that ebastine might be useful for the treatment of T cell-mediated allergic inflammatory disorders, including asthma, atopic dermatitis, and Th2-type autoimmune diseases.     

Th1/Th2 cytokines reciprocally regulate in vitro pulmonary angiogenesis via CXC chemokine synthesis

Hypervascularity is known as an important element of airway remodeling in bronchial asthma. However, it remains obscure how allergic inflammation relates to angiogenesis in the lung. In this study, we examined the in vitro effects of inflammatory cytokines on endothelial cell functions, particularly angiogenesis. Human microvascular endothelial cells from normal lung (HMVEC-Ls) were cultured with TNF-alpha, IFN-gamma, IL-4, a combination of TNF-alpha and IFN-gamma, or a combination of TNF-alpha and IL-4, and the cell proliferation and tube-forming activities were evaluated. IL-4 slightly enhanced the proliferation of HMVEC-Ls in the presence of vascular endothelial growth factor (VEGF), whereas TNF-alpha and IFN-gamma tended to inhibit it. Synergistic inhibition was observed when TNF-alpha and IFN-gamma were simultaneously added to the culture medium. The combination of IL-4 and TNF-alpha markedly promoted tube formation by HMVEC-Ls, even in the absence of VEGF. The IL-4 and TNF-alpha combination induced autocrine production of CXCR2 chemokines, which are known to have angiogenic activity, whereas the production of angiostatic CXCR3 chemokines was dramatically up-regulated when TNF-alpha and IFN-gamma were present. The marked IL-4- and TNF-alpha-induced tube formation was inhibited by a selective CXCR2 antagonist. These results suggest that, in the presence of TNF-alpha, IL-4 and IFN-gamma reciprocally regulate tube formation by HMVEC-Ls through autocrine synthesis of CXCR2 and CXCR3 chemokines, respectively. Of note, the CXCR2 chemokine-induced tube formation was independent of VEGF. Therefore, CXCR2 chemokines may represent potential therapeutic targets for bronchial asthma.     

Modulation of human microglia and THP-1 cell toxicity by cytokines endogenous to the nervous system

Neuroinflammatory processes are thought to be a significant factor in the pathology of a number of degenerative neurological diseases. A variety of cytokines influence inflammatory levels. Here we show that a cooperative action of two or more cytokines is required to induce significantly human microglial and monocytic THP-1 cell toxicity towards SH-SY5Y neuroblastoma cells. Such toxicity was induced by the following combinations: interferon-gamma (IFN-gamma) with tumor necrosis factor-alpha (TNF-alpha); IFN-gamma with interleukin (IL) 1alpha or IL-1beta in the presence of TNF-alpha; and IL-6 with TNF-alpha. Toxicity induced by the various stimulatory combinations was not accompanied by an increased nitrite production. Of the potential inhibitors tested, IL-4 downregulated the toxic action of microglia when applied to THP-1 cells either before stimulation or 24 h after stimulation. Toxicity was not inhibited by IL-10, and was even enhanced by transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF). These data suggest that antagonists of cytokine receptors, as well as inhibitors of their intracellular pathways may be effective anti-inflammatory agents.     

Inefficient protection of human TAP-deficient fibroblasts from autologous NK cell-mediated lysis by cytokines inducing HLA class I expression

We studied HLA class I expression and susceptibility to lysis of activated autologous NK cells in normal and TAP-deficient fibroblasts. These cells were cultured in the presence or absence of cytokines known to increase the surface expression of HLA class I molecules. All the cytokines tested (IFN-alpha, IFN-gamma, TNF-alpha and IFN-gamma + TNF-alpha) increased the expression of HLA class I molecules on fibroblasts after 48-h culture, but on TAP-deficient cells this expression remained very low as compared to that of normal cells. In the presence of IFN-alpha, IFN-gamma or IFN-gamma + TNF-alpha, normal target cells became resistant to lysis by autologous NK cells, whereas this effect was much less pronounced in the case of TAP-deficient fibroblasts. Addition of an anti-HLA class I mAb to fibroblasts treated with cytokines increased lysis of normal but not of TAP-deficient cells. These results suggest that activated TAP-deficient NK cells are strongly cytotoxic to normal autologous cells and that these cells cannot be efficiently protected by cytokines inducing HLA class I expression. Thus, in human TAP deficiency, activated NK cells may contribute to the progressive lung degradation which characterizes the clinical course of these patients.     

Cytokine sensitivity and methylation of lysine in Rickettsia prowazekii EVir and interferon-resistant R. prowazekii strains.

Modified Rickettsia prowazekii strains have been derived from the avirulent Madrid E strain by passage in the lungs of white mice (strain EVir) or by selection for resistance to gamma interferon (IFN-gamma) (strains 427-19 and 87-17) or alpha/beta interferon (IFN-alpha/beta) (strains 83-2P, 60P, 103-2P, and 110-1P). Compared with the Madrid E strain, strain EVir has increased virulence (N. M. Balayeva and V. N. Nikolskaya, J. Hyg. Epidemiol. Microbiol. Immunol. 17:11-20, 1973) and a different lysine methylation profile in its surface protein antigen (A. V. Rodionov, M. E. Eremeeva, and N. M. Balayeva, Acta Virol. 35:557-565, 1991). The other six strains differ from the Madrid E strain in their resistance to IFN and their ability to grow well in untreated macrophagelike RAW264.7 cells. In the present study, to determine which properties are shared by these strains, we examined R. prowazekii EVir for the following: (i) the sensitivity of its growth in L929 cells to the cytokines IFN-alpha/beta, IFN-gamma, tumor necrosis factor alpha (TNF-alpha), and IFN-gamma plus TNF-alpha; (ii) the ability to grow in untreated RAW264.7 cells; and (iii) the ability to induce interferon in L929 cell cultures; we also evaluated strains 83-2P and 87-17 for lysine methylation. Multiplication of strain EVir in growing L929 cells was not markedly inhibited by either IFN-alpha/beta or IFN-gamma. In X-irradiated L929 cells, growth of strain EVir was slightly inhibited (11%) by TNF-alpha alone, somewhat inhibited (38%) by IFN-gamma alone, and markedly inhibited (87%) by IFN-gamma plus TNF-alpha. Nitrite production was induced in X-irradiated, strain EVir-infected L929 cell cultures treated with TNF-alpha alone or IFN-gamma alone; however, more nitrite was produced in infected cultures treated with IFN-gamma plus TNF-alpha. Nitrite production, the dramatic inhibitory effect of IFN-gamma plus TNF-alpha, and the modest inhibitory effect of IFN-gamma on the growth of strain EVir in X-irradiated L929 cells were all alleviated by the addition of the nitric oxide synthase inhibitor NG-methyl-L-arginine. Strain EVir grew very well in untreated macrophagelike RAW264.7 cells and appeared defective in the ability to induce IFN in L929 cell cultures. All strains grown in L929 cells in the presence of radiolabeled lysine had similar percentages of their radioactivity as methylated lysines.(ABSTRACT TRUNCATED AT 400 WORDS)     

Benzylpenicillin differentially conjugates to IFN-gamma,TNF-alpha,IL-1beta,IL-4 and IL-13 but selectively reduces IFN-gamma activity

It is known that beta-lactam antibiotics can conjugate to lysine and histidine residues on proteins via the carbonyl group of the opened beta-lactam ring. However, it is not known which proteins these drugs target and there is little work addressing whether conjugation is preferential for some proteins over others or if conjugation has functional consequences for the protein. We have previously shown that the beta-lactam antibiotic benzylpenicillin (BP) conjugates to IFN-gamma and reduces its activity. This interaction demonstrates selectivity, as BP does not bind to IL-4. Here, we extend our study to include other Th1 and Th2 cell-associated cytokines and two cytokines associated with inflammatory responses. We demonstrate by Western blotting that BP also conjugates to IL-1beta, IL-2, IL-5, IL-13 and TNF-alpha but not to IL-10. Densitometric analysis of leading cytokine bands on blots revealed that IFN-gamma always gave more intense BP-positive bands than any other cytokine analysed. Cytokines pre-incubated with BP at 37 degrees C in a protein-containing, serum-free medium were assayed for their biological activity. By in vitro bioassay, BP inhibited the ability of IFN-gamma but not IL-1beta or TNF-alpha to induce CD54 expression on epithelial cells. In addition, BP did not affect IL-4 or IL-13 inhibition of mast cell proliferation. When the pre-incubation temperature was reduced to 4 degrees C, BP did not conjugate to IFN-gamma or modulate its activity. BP retained its inhibitory effect on IFN-gamma activity when 20% FCS was added to the pre-incubation medium. In conclusion, BP conjugates to some cytokines but not others and this does not appear to be related to primary protein structure. Furthermore, of the cytokines studied, conjugation only to IFN-gamma is accompanied by inhibition of activity. This phenomenon is temperature dependent and occurs in the presence of serum. These findings provide further evidence for differential, direct drug-cytokine interactions. Such interactions may have therapeutic implications in terms of targeting cytokines to regulate their activity.     

Interferon-gamma enhances rhinovirus-induced RANTES secretion by airway epithelial cells

Respiratory viruses, including rhinoviruses, infect respiratory epithelium and induce a variety of cytokines and chemokines that can initiate an inflammatory response. Cytokines, such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, could enhance epithelial cell activation by inducing virus receptors. To test this hypothesis, effects of IFN-gamma or TNF-alpha on expression of intercellular adhesion molecule (ICAM)-1, rhinovirus binding, and virus-induced chemokine secretion on A549 and human bronchial epithelial cells (HBEC) were determined. The results varied with the type of cell. IFN-gamma was a stronger inducer of ICAM-1 and viral binding on HBEC, whereas TNF-alpha had greater effects on A549 cells. In addition, IFN-gamma, but not TNF-alpha, synergistically enhanced regulated on activation, normal T cells expressed and secreted (RANTES) mRNA expression and protein secretion induced by RV16 or RV49. To determine whether IFN-gamma could enhance RANTES secretion independent of effects on ICAM-1 and RV binding, HBEC were transfected with RV16 RNA in the presence or absence of IFN-gamma. RV16 RNA alone stimulated RANTES secretion, and this effect was enhanced by IFN-gamma. These results demonstrate that IFN-gamma can enhance rhinovirus-induced RANTES secretion by increasing viral binding, and through a second receptor-independent pathway. These findings suggest that IFN-gamma, by upregulating RANTES secretion, could be an important regulator of the initial immune response to rhinovirus infections.     

Effects of interferon-gamma and tumor necrosis factor-alpha on clonogenic growth of cell lines and primary cultures from human gliomas and brain metastases

In this study we tested the effect of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and a combination of these immunotherapeutics on clonogenic growth of cell lines and primary cultures of malignant human brain tumors. Out of 29 primary cultures studied, 5 were inhibited 50% or more by IFN-gamma (10 ng/ml), 6 were inhibited 50% or more by TNF-alpha (10 ng/ml) and 14 were inhibited 50% or more by a combination of IFN-gamma (10 ng/ml) and TNF-alpha (10 ng/ml). The doses of the immunotherapeutics used in this in vitro study are achievable in serum after intravenous administration of IFN-gamma and TNF-alpha without causing unacceptable side effects. We believe that some glioma patients and some patients with brain metastases will benefit from receiving treatment with IFN-gamma, TNF-alpha or a combination of these immunotherapeutics. The patients should be selected for treatment with these immunotherapeutics according to in vitro sensitivity results.     

Role of the nitric oxide synthase pathway in inhibition of growth of interferon-sensitive and interferon-resistant Rickettsia prowazekii strains in L929 cells treated with tumor necrosis factor alpha and gamma interferon.

The ability of tumor necrosis factor alpha (TNF-alpha) alone and in combination with gamma interferon (IFN-gamma) to inhibit the growth of interferon-sensitive and -resistant Rickettsia prowazekii strains in mouse L929 cells was examined, and the possible role of the nitric oxide synthase pathway in the suppression of rickettsial growth induced by TNF-alpha, IFN-gamma, or both cytokines was evaluated. TNF-alpha inhibited the growth of strains Madrid E (IFN-gamma sensitive and alpha/beta interferon [IFN-alpha/beta] sensitive) and Breinl (IFN-gamma sensitive and IFN-alpha/beta resistant), but not that of strain 83-2P (IFN-gamma resistant and IFN-alpha/beta resistant), in L929 cells. Inhibition of the growth of the Madrid E strain in L929 cells treated with TNF-alpha and IFN-gamma in combination was greater than that observed with either TNF-alpha or IFN-gamma alone. Similarly, inhibition of the growth of the Breinl strain in L929 cells treated with both cytokines was greater than that observed with TNF-alpha alone; however, it did not differ significantly from the inhibition observed with IFN-gamma alone. Although strain 83-2P was resistant to TNF-alpha or IFN-gamma alone, its growth was inhibited in L929 cells treated with TNF-alpha and IFN-gamma in combination. Nitrite production was measured in mock-infected and infected L929 cell cultures, and the nitric oxide synthase inhibitors NG-methyl-L-arginine (NGMA) and aminoguanidine were used to evaluate the role of the nitric oxide synthase pathway in cytokine-induced inhibition of rickettsial growth. Nitrite production was induced in mock-infected or R. prowazekii-infected L929 cell cultures treated with IFN-gamma plus TNF-alpha, but not in mock-infected cultures that were untreated or treated with IFN-gamma or TNF-alpha alone. Nitrite production was also not induced in untreated, R. prowazekii-infected cultures; however, in some instances, it was induced in infected cultures treated with IFN-gamma or TNF-alpha alone. Nitrite production was blocked by NGMA or aminoguanidine, and these compounds markedly relieved the synergistic inhibitory effect of IFN-gamma plus TNF-alpha on the growth of strain 83-2P in L929 cells. In contrast, NGMA did not alleviate the inhibition of the growth of the Madrid E strain in L929 cells treated with IFN-gamma or TNF-alpha alone; however, it slightly and variably relieved the inhibition of the growth of the Madrid E strain in L929 cells treated with IFN-gamma and TNF-alpha in combination.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of E-cadherin, alpha- & beta-catenin, and CD44V6 and the subcellular localization of E-cadherin and CD44V6 in normal epidermis and basal cell carcinoma

Basal cell carcinoma (BCC) of the skin is a locally invasive, rarely metastasizing epithelial tumor. In the current study, the expression of E-cadherin, alpha- and beta-catenin and CD44V6 in normal epidermis and on BCC cells were investigated. A significantly reduced expression of alpha-catenin and CD44V6 and a slightly reduced expression of E-cadherin on BCC cells were observed compared with the overlying epidermis. Immunoelectron microscopy was used to investigate whether the decreased expression of E-cadherin and CD44V6 was due to either an absence or downregulation of specific membrane structures or due to an overall downregulation of these adhesion molecules in all membrane structures in BCC. E-cadherin and CD44V6 were expressed in adherens junctions, desmosomes, and complex interdigitating membrane structures both in normal epidermis and in BCC. A quantitative analysis showed that only a percentage of desmosomes was stained. In addition, the effect of pro-inflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), was investigated in biopsy specimens of normal skin and BCC, using a biopsy culture system and immunohistochemistry. The expression of E-cadherin and CD44V6 was not significantly decreased after culturing BCC or normal skin biopsy specimens for 48 hours with or without recombinant human (rHu)IFN-gamma or rHuTNF-alpha. It may be concluded that the decreased expression of both E-cadherin and CD44V6, observed in light microscopy, was not attributable to the absence of specific specialized structures in BCC and most likely also not caused by downregulation by local cytokines, but rather by generic downregulation of both of these adhesion molecules during malignant transformation.     

Effects of associated cytokines (IL-1, TNF-alpha, IFN-gamma and TGF-beta) on collagen and glycosaminoglycan production by cultured human synovial cells

The production of collagen and glycosaminoglycans (GAG) was studied in cultured human synovial cells exposed to four cytokines, alone or in dual combination, namely interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). Among these cytokines, only TGF-beta (0.1-10 ng/ml) induced a significant and dose-dependent increase of collagen synthesis in a 24-h incubation. This effect was reversed when the factor was associated with either IL-1 beta (100-500 pg/ml), TNF-alpha (1-100 ng/ml) or IFN-gamma (100 U/ml). Except IFN-gamma which clearly inhibits the collagen production, the other cytokines IL-1 and TNF-alpha were not very effective when tested separately, although they generally induced a small reduction in collagen amount. IL-1 beta and TNF-alpha were found to be more efficient than TGF-beta in stimulating the production of GAG by the synovial cells. IFN-gamma exerted an antagonistic effect on the TGF-beta-induced stimulation of GAG synthesis. TNF-alpha and IL-1 beta were shown to have an additive effect on that production. The results indicate that interactions between cytokines present in the inflamed synovial tissue may modulate their respective actions and thus introduce differentials in their effect on collagen and GAG metabolism which are responsible for the alterations of synovial extracellular matrix in rheumatoid arthritis.     

Interferon-mediated block in cell cycle and altered integrin expression in a differentiated salivary gland cell line (HSG) cultured on Matrigel.

Sj?gren's syndrome (SS), an idiopathic, autoimmune exocrinopathy, is partly characterized by diminished salivary flow, acinar cell atrophy, and increased expression of several cytokines. Several in vivo characteristics of the sialoadenitis are also evident in a human salivary gland ductal epithelial cell line (HSG) treated with cytokines. HSG cells differentiate to the acinar phenotype when cultured on Matrigel (Becton Dickinson, Bedford, MA), a basement membrane extract. To elucidate mechanisms of salivary gland pathology, the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on cell cycle progression and integrin expression were evaluated in HSG acinarlike cells. Flow cytometry experiments showed that cytokine treatment for 2 days arrested cells in G(1) phase of the cell cycle, and this preceded significant morphologic changes and decreased viability. Whereas only modest cytokine-mediated increases in protein expression for the alpha 3 and beta 1 integrin subunits were seen by immunoprecipitation, a form of alpha 3 integrin displaying enhanced electrophoretic mobility was evident after 6 days of cytokine treatment. To our knowledge, this is the first report demonstrating an IFN-mediated alteration in the electrophoretic mobility of integrin subunits. From this study, it was evident that the combination of IFN-gamma and TNF-alpha resulted in a block in G(1) phase for acinar cells before accumulation of the alpha 3 integrin variant or significant degenerative cellular changes. Information from the present and previous studies suggests that cytokines may alter the pattern of integrin expression and block cell cycle progression in salivary gland cells grown in three-dimensional acinarlike clusters. These experiments may provide a new cell culture model to study the effects of cytokines in normal and diseased salivary glands, including SS.     

Expression of lymphotoxin-alpha by keratinocytes: a further mediator for the lichenoid reaction.

OBJECTIVE: Lichen planus (LP) represents a disease in which autoimmune mechanisms mediated by Th1 T cells are involved. Lymphotoxin-alpha (LT-alpha) represents a Th1 cytokine with proinflammatory activities in LP, as has recently been demonstrated for interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). METHODS: Expression of LT-alpha mRNA was investigated by RT-PCR and nonradioactive in situ hybridization. Double staining methods were applied to characterize the phenotype of cells expressing LT-alpha. Cell stimulation experiments were performed on the transformed squamous cell line HaCaT. RESULTS: In contrast to normal skin, LT-alpha-specific RT-PCR products were found in all cases of LP. Cells in the inflammatory infiltrate expressing LT-alpha were identified as mainly T cells and mast cells, as shown by in situ hybridization. Furthermore, predominant LT-alpha mRNA expression could be observed in lesional keratinocytes adjacent to the band-like inflammatory infiltrate. In cell stimulation experiments, it could be shown that IFN-gamma induces LT-alpha and TNF-alpha mRNA in the human squamous cell line HaCaT, concomitant with upregulation of MHC class II and intercellular adhesion molecule-1, which could also be observed on lesional keratinocytes in LP. CONCLUSIONS: In LP, LT-alpha mRNA is predominantly expressed by lesional keratinocytes and to a lesser extent by inflammatory cells. Induction of LT-alpha in keratinocytes is closely related to the expression of TNF-alpha and MHC class II. The loci of TNF-alpha and LT-alpha map to MHC class III on chromosome 6, which is closely linked to the MHC class II gene locus. Our results suggest that stimulation of keratinocytes with IFN-gamma results in the upregulation of proinflammatory cytokines such as LT-alpha and TNF-alpha as well as MHC class II, which map to the same gene region of immunoregulatory genes on chromosome 6 and may be involved in the induction and maintenance of the disease.     

Reduced cytokine secretions by LAK cells of pulmonary tuberculosis patients in response to tumor targets in vitro.

Activation of macrophages and other immune components to release a series of proinflammatory cytokines is one of the first events in innate resistance to intracellular infections. Severe manifestations of tuberculosis (TB) could be caused by alterations in the balance of these cytokines. In this study, lymphokine-activated killer (LAK) cells of TB patients and normal individuals were generated by stimulation with cytokines in vitro. The LAK cells of both groups were further triggered with allogeneic tumor targets. Cytokines interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were estimated in the supernatants generated in the two groups. The aim was to see if infection with TB influenced the secretory capacity of the immune cells in vitro. Reduced cytokine profiles were observed in TB patients, indicating defective interactions between patient effector cells with allogeneic transformed cells compared with normal individuals. Partial restoration of IFN-gamma production was seen with a combination of cytokines interleukin-2 (IL-2) and IL-12 in TB patients. Based on the in vitro observations, we hypothesize that in vivo also there is diminished immune cell activation of effector cells in response to the presence of infected macrophages. This probably leads to a diminished secretory function that can be corrected by the use of such cytokines as IL-2 and IL-12. The effector populations of TB patients are probably in a state of target-induced anergy, allowing the bacteria to thrive, and immunomodulatory cytokines that improve the host immune response toward countering mycobacterial infection.