Search

Objective: To identify the role of furin, TNF-α, and TGF-β2 in human missed abortion pathogenesis., Study Design: Decidual materials were collected from patients diagnosed with a missed abortion (n = 10) (missed abortion group) and from legal voluntary termination cases at < 10 gestational weeks (n = 10) (normal pregnancy group). Tissue samples were collected from each group by dilation and curettage under mask anesthesia. For all tissue samples,furin, TNF-α, and TGF-β2 primary antibodies were performed by immunohistochemical staining. The number of stained cells was evaluated by using the H-score technique., Results: In immunohistochemical examination, the immunoreactivities of furin, TNF-α, and TGF-β2 were found to be higher in syncytiotrophoblastic cells in the missed abortion group than in the normal pregnancy group (p < 0.005). Additionally, high immunoreactivity of TNF-α and TGF-β2 molecules was established only in cytotrophoblastic cells of missed abortions (p < 0.005) in examination at decidual cells of the missed abortion group; furin immunoreactivities were detected higher in the missed abortion group than in the control group, but TNF-α and TGF-β2 immunoreactivity were increased in number in the normal pregnancy group (p < 0.005)., Conclusion: It is considered that high levels offurin and the 2 furin-related proteins (TNF-α and TGF-β2), which play important roles in proliferation, invasion, migration, differentiation, and survival of cells, may be the reason of proceeding decidualization, placentation, and prevention from abortion, in spite of terminating thefetal life.

Tumor necrosis factor (TNF) is one of the most important proinflammatory cytokines. It demonstrates a complex pattern of tissue-specific expression and behaves as a product of immediate early transcriptional response in macrophages. These properties have made the regulation of TNF gene, as well as regulation of tightly linked related lymphotoxin alpha (LTalpha) and beta (LTbeta) genes the object of thorough investigation for more than two decades. Some aspects of TNF/LT locus regulation, such as the role of distal TNF-promoter and of NF-kappaB factors in TNF gene transcription, still remain the object of discussion. Moreover, several recent studies uncovering the molecular mechanisms of immediate early gene activation and directly related to TNF gene regulation have not been reflected in published reviews yet. Here we briefly overview the modern concepts of transcriptional regulation of the TNF/LT locus, with an accent on new data and unanswered questions.

Lymphotoxin-beta (LT-beta) is a transmembrane protein expressed mainly on cells of the lymphoid lineage. It associates with LT-alpha on the cell surface to form the heterotrimeric LTalpha1,beta2 complex, which binds the LT-beta receptor. Membrane lymphotoxin is a crucial signal for the appropriate development of lymph nodes and Peyer's patches, and in the formation of B and T cell compartments in the spleen. In this study we report the characterization of mechanisms governing both basal as well as PMA- and TNF-inducible regulation of the human LT-beta promoter. Using a Jurkat T cell line, induction with either PMA or TNF resulted in an increase in mRNA levels compared with uninduced values. This induction corresponded to an increase in transcriptional activity of the human LT-beta promoter. Mutational and deletion analysis demonstrated the importance of Ets and NF-kappaB motifs in the regulation of basal transcription. Furthermore, the ability of PMA to induce activity was lost in the Ets mutant constructs. Interestingly, the same mutation had little effect on the ability of TNF to induce transcription of the LT-beta promoter. TNF inducibility was localized to the NF-kappaB site positioned at -83 of the promoter sequence. Thus, it appears that the Ets site, although playing a major role in PMA induction, did not mediate TNF inducibility. Therefore, our study suggests that alternative signaling pathways may be present to induce the expression of LT-beta in response to different immunological or inflammatory stimuli.

Significantly higher frequencies of tumor necrosis factor alpha- and interleukin-2-secreting human T-lymphotropic virus type 1 (HTLV-1)-specific CD4(+) T cells were present in the peripheral blood mononuclear cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients than in those of asymptomatic carriers with similar provirus loads. The data suggest that HTLV-1-specific CD4(+) T cells play a role in the pathogenesis of HAM/TSP.

The heteromeric lymphotoxin alphabeta ligand (LT) binds to the LTbeta receptor (LTbetaR) and provides an essential trigger for lymph node (LN) development. LTbetaR signaling is also critical for the emergence of pathological ectopic lymph node-like structures and the maintenance of an organized splenic white pulp. To better understand the role of LT in development, the expression patterns of LTbeta and LTbetaR mRNA were examined by in situ hybridization in the developing mouse embryo. Images of LTbeta ligand expression in developing peripheral LN in the E18.5 embryo revealed a relatively early phase structure and allowed for comparative staging with LN development in rat and humans. The LTbetaR is expressed from E16.5 onward in respiratory, salivary, bronchial, and gastric epithelium, which may be consistent with early communication events between lymphoid elements and epithelial specialization over emerging mucosal LN. Direct comparison of mouse fetal and adult tissues by FACS analysis confirmed the elevated expression of LTBR in some embryonic epithelial layers. Therefore, surface LTBR expression may be elevated during fetal development in some epithelial layers.

Lymphotoxin-beta (LT- beta) is a tumor necrosis factor (TNF)-related membrane-bound cytokine that forms a heterotrimeric surface lymphotoxin (LT) complex with LT-alpha on the surface of lymphoid cells. Although knockout studies have revealed a role in lymph node biogenesis during development, the regulation and function of surface LT in mature cell types are poorly understood. The present study aims to understand the physiologic signals that regulate the components of surface LT in Jurkat T cells. We show that the previously observed upregulation of surface LT by phorbol myristate acetate (PMA) is markedly abrogated by cotreatment with ionomycin through posttranscriptional mechanisms. In addition, the observation of striking similarities between the mRNA accumulation kinetics of LT-alpha and LT-beta during these treatments indicates tight coupling of expression under certain conditions. In investigating the reported upregulation of LT-beta during inflammation, we tested the effects of various proinflammatory and anti-inflammatory cytokines on LT-beta expression. Our data demonstrate an upregulation of LT-beta mRNA by the inflammatory cytokines TNF and LT-alpha.

IL-2 induces the stimulation of inflammatory and immune reactions, and the apoptosis of antigen-activated cells. However, the molecular basis of these pleiotropic functions is largely unknown. We have previously reported that IL-2 induces genes involved in cytoskeleton organization, oncogene regulation and transcriptional control. In an IL-2-dependent cell line, we have also shown that IL-2 induces tumor necrosis factor (TNF)-beta mRNA through the Jak-STAT pathway. Here, we first demonstrate in vitro that IL-2 induces mature and partially spliced TNF-beta mRNA in the splenocytes and lymph node cells of both IL-2(-/-) and IL-2(+/-) mice. Under the same experimental conditions, IL-2 is seen to induce TNF-alpha mRNA. mRNA expression is followed by semiquantitative RT-PCR and this analysis is then extended in vivo by studying different lymphoid organs from IL-2(-/-)animals. Strikingly, the expression of TNF-beta mRNA is noted to be extremely low in the spleens and lymph nodes of IL-2(-/-) mice. Similarly, TNF-alpha, lymphotoxin (LT)-beta, TNFR1 and TNFR2 mRNA levels are also low in the spleens of IL-2(-/-) animals, whereas IFN-gamma and IL-4 mRNA levels remain unaffected in these animals. The experimental values are significantly different (P < or = 0.05) from those of control IL-2(+/-) animals. Western blot analysis of TNF-alpha expression confirmed and extended the results at the protein level. For the first time, we demonstrate that IL-2 directly or indirectly regulates genes of the TNF-TNFR family in secondary lymphoid organs. Furthermore, IL-2(-/-) animals in which thymopoiesis is unaffected show normal expression of these genes. Altogether, our data further define the pleiotropic effects of IL-2 at the molecular level.

The formation of germinal centers (GC) around follicular dendritic cells (FDC) is a critical step in the humoral immune responses that depends on the cooperative effects of B cells and T cells. Mice deficient in either TNF or lymphotoxin (LT) fail to form both GC and FDC network in B cell follicles. To test a potential complementary effect of TNF and LT, a mixture of bone marrow cells from TNF(-/-) mice and LT alpha(-/-) mice was transferred into irradiated LT alpha(-/-) mice or TNF(-/-) mice. Interestingly, the formation of both GC and FDC clusters in B cell follicles was restored in such chimeric mice, suggesting that TNF and LT from different cells could complement one another. To identify the exact contributions of each subset to the complementary effect of TNF and LT, different sources of T and B cells from LT alpha(-/-) mice or TNF(-/-) mice were used for reconstitution. Our study demonstrates that either T or B cell-derived TNF is sufficient to restore FDC/GC in the presence of LT-expressing B cells. However, TNF itself is not required for GC reactions if the FDC network is already intact. Thus, the development and maintenance of these lymphoid structures depend on a delicate interaction between TNF and LT from different subsets of lymphocytes.

Excessive production of the tumor necrosis factor (TNF) ligand-receptor system has been found to contribute to the severity of non-Hodgkin's lymphoma (NHL). We therefore investigated the expression of TNF, lymphotoxin alpha (LTalpha), lymphotoxin beta (LTbeta), and their receptor (p55, p75, LTbeta-R) transcripts within the tumor tissue in different NHL histological subtypes. The constitutive expression of genes coding for TNF-related ligands and receptors was found in almost all 31 NHL samples studied. Semi-quantitative reverse transcription/polymerase chain reaction and computed densitometry assays revealed that the amounts of TNF, LTalpha, p55, and LTbeta-R mRNA were higher in follicular NHL than in other histological entities. Therefore tumor cell immunopurification was performed in representative follicular NHL samples and consistent results were obtained. The pattern of LTbeta gene expression was different from that of the other molecules, indicating the existence of distinct mechanisms of gene regulation. These results indicate that the transcription of genes coding for the TNF ligand-receptor system in NHL tumor tissue is more widespread than originally thought and that the heterogeneity of their expressions might be related to histological features. The expression of TNF-related ligands and receptors in tumor tissues is likely to contribute to the clinicopathological features of lymphoid-derived malignancies.

Purpose: To determine the effect of polymorphic variations in the tumor necrosis factor alpha (TNFalpha) and lymphotoxin alpha (LTalpha) genes on the predisposition to myeloma and the effect of these polymorphisms on response to treatment and overall survival., Patients and Methods: Genotype distribution was determined in 63 patients with monoclonal gammopathy of uncertain significance (MGUS) and 198 patients with myeloma and compared with that in 250 age- and sex-matched population-based controls. The effect on treatment response and survival was determined in 171 myeloma patients treated with either conventional or high-dose chemotherapy., Results: Comparison of the extended TNFalpha/LTalpha haplotype in the myeloma cases and controls showed a significant excess of high-producer alleles in the cases. The double heterozygotes TNF1/2 and LT10.5/5.5 were present in 35.8% of cases but in only 18% of the controls; this presence was associated with a significant increased risk of myeloma (odds ratio, 2.05; 95% confidence interval, 1.26 to 3.35). A similar odds ratio was seen in the MGUS cases, suggesting that this genotype is associated with the initiation of plasma-cell disorders rather than the progression of MGUS to myeloma. The median overall survival time of myeloma patients was 53.8 months and showed no difference with regard to TNFalpha/LTalpha polymorphic status. A trend toward an improved progression-free survival was apparent in cases with a high-producer haplotype, although this effect was seen only in patients receiving high-dose chemotherapy., Conclusion: Individuals with polymorphisms associated with a high production of TNFalpha/LTalpha are at a significantly increased risk of developing MGUS and myeloma. The impact of polymorphic status on overall survival is minimal, although there is a trend toward an increased progression-free survival in the high-producer group.

The receptor for interleukin-6 (IL-6) consists of two subunits: a ligand specific IL-6Ralpha and gp130 that is responsible for signal-transduction. A soluble form of the ligand specific chain was described that when complexed to IL-6 is capable of binding to the membrane-bound gp130 subunit and thus can elicit signal-transduction. This soluble receptor can act on cells that express only the gp130 but not the ligand-specific subunit of the IL-6R. This phenomenon, called trans-signaling, introduced a novel aspect of cytokine action. In this study we examined the response of Jurkat cells, that are known not to express IL-6Ralpha, to IL-6, the soluble IL-6 receptor (sIL-6R) and a covalent complex of IL-6 and sIL-6R termed Hyper-IL-6. We studied the expression of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). The complex of IL-6+sIL-6R and Hyper-IL-6 inhibited significantly the production of TNF in a gp130-dependent manner, whereas no differences in IFN-gamma expression were found. IL-6 and sIL-6R alone were not effective. Because we did not detect major differences in the TNF mRNA levels upon treatments, we conclude that the inhibition of TNF production should occur at the post-transcriptional level. These results provide another example of trans-signaling and underline the physiological importance of sIL-6R, and in the case of Hyper-IL-6 its possible therapeutic application can also be considered.

An interferon-gamma (IFN-gamma)-inducing molecule (OK-PSA) has been purified from OK-432 by an affinity chromatographic technique performed on cyanogen bromide-activated Sepharose 4B-bound TS-2 monoclonal antibody, which neutralizes IFN-gamma-inducing activity of OK-432. OK-PSA has striking anti-tumor activity in vivo and in vitro. In the current study, the liposomes were used to improve the delivery of the agent (OK-PSA) to effector cells and to increase the therapeutic effect. Significantly less OK-PSA encapsulated into liposomes (Lipo-OK-PSA) than OK-PSA alone (1/100 or less of OK-PSA alone) was required to induce IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), TNF-beta, interleukin-1 beta (IL-1 beta), natural killer, and lymphokine-activated killer activities by human peripheral blood mononuclear cells and mouse spleen cells. Furthermore, higher levels of these activities were detected in peripheral blood mononuclear cells and mouse spleen cells treated with Lipo-OK-PSA than in those treated with OK-PSA. All of these activities induced by Lipo-OK-PSA were almost completely neutralized by anti-asialo-GM1 antibody and complement (p < 0.001). In in vivo experiments, Lipo-OK-PSA elicited striking anti-tumor activity on syngeneic Meth-A tumor-bearing and colon 26-bearing BALB/c mice and on salivary gland tumor-bearing nude mice far better than did OK-PSA. Furthermore, high levels of natural killer and lymphokine-activated killer activities and a significant increase in the number of cells positive for asialo-GM1, IFN-gamma, TNF-alpha, or IL-1 beta were detected in the spleen cells derived from the animals given Lipo-OK-PSA compared with those given saline. These findings clearly indicate that OK-PSA plays an important role in the anti-tumor efficiency of OK-432, and that, for the most part, liposome encapsulation of this molecule markedly accelerates its effect mediated by asialo-GM1-positive cells (mainly natural killer cells).

Natural killer cells mediate spontaneously secretory/necrotic killing against rare leukemia cell lines and a nonsecretory/apoptotic killing against a large variety of tumor cell lines. The molecules involved in nonsecretory/apoptotic killing are largely undefined. In the present study, freshly isolated, nonactivated, human NK cells were shown to express TNF, lymphotoxin (LT)-alpha, LT-beta, Fas ligand (L), CD27L, CD30L, OX40L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), but not CD40L or nerve growth factor. Complementary receptors were demonstrated to be expressed on the cell surface of solid tumor cell lines susceptible to apoptotic killing mediated by NK cells. Individually applied, antagonists of TNF, LT-alpha1beta2, or FasL fully inhibited NK cell-mediated apoptotic killing of tumor cells. On the other hand, recombinant TNF, LT-alpha1beta2, or FasL applied individually or as pairs were not cytotoxic. In contrast, a mixture of the three ligands mediated significant apoptosis in tumor cells. These findings demonstrate that human NK cells constitutively express several of the TNF family ligands and induce apoptosis in tumor cells by simultaneous engagement of at least three of these cytotoxic molecules.

Dengue hemorrhagic fever, the severe form of dengue virus infection, is believed to be an immunopathological response to a secondary infection with a heterologous serotype of dengue virus. Dengue virus capsid protein-specific CD4(+) cytotoxic T-lymphocyte (CTL) clones were shown to be capable of mediating bystander lysis of non-antigen-presenting target cells. After activation by anti-CD3 or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Lysis of HepG2 cells suggests a potential role for CD4(+) CTL in the liver involvement observed during dengue virus infection. Three CD4(+) CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/Fas ligand interactions. In contrast, one clone used a Fas/Fas ligand mechanism to lyse both cognate and bystander targets. Cytokine production by the CTL clones was also examined. In response to stimulation with D2 antigen, CD4(+) T-cell clones produced gamma interferon, tumor necrosis factor alpha (TNF-alpha) and TNF-beta. The data suggest that CD4(+) CTL clones may contribute to the immunopathology observed upon secondary dengue virus infections through direct cytolysis and/or cytokine production.

Lymphotoxin (LT) is a cytokine that orchestrates lymphoid neogenesis and formation of germinal center reactions. LT exists as a membrane heterotrimer of alpha and beta subunits and is secreted as a homotrimer, LTalpha3. Using LTbetaR.Fc, expression of LTalphabeta on CD4 T cell subsets was investigated in a TCR transgenic model. LTalphabeta was evident 24-72 h after activation of naive T cells with specific Ag, and declined thereafter. Early expression was independent of IFN-gamma and IL-12, however, IL-12 prolonged expression. LTalphabeta was reinduced within 2-4 h after Ag restimulation, but declined by 24 h regardless of IL-12 or IFN-gamma priming. Exposure of naive T cells to IL-4 did not affect early LTalphabeta expression at 24 h, but resulted in subsequent down-regulation. IL-4-differentiated Th2 effectors did not re-express LTalphabeta, and LTalphabeta was transiently found on Th1 clones but not Th2 clones. LTalpha3 and TNF were immunoprecipitated from supernatants and lysates of IL-12 primed cells but not IL-4 primed cells. These studies demonstrate that LTalphabeta is expressed by activated naive CD4 cells, unpolarized IL-2-secreting effectors, and Th1 effectors. In contrast, loss of surface LTalphabeta and a lack of LTalpha3 and TNF secretion is associated with prior exposure to IL-4 and a Th2 phenotype.

Osteoprotegerin (OPG) is a recently cloned soluble member of the tumor necrosis factor receptor family. OPG has been shown to inhibit osteoclast recruitment by binding to OPG-ligand, an osteoclast differentiating factor on osteoblastic stromal cells, thereby blocking osteoclastogenesis. In this report we have examined the effect of tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta) on OPG mRNA levels in the human osteosarcoma cell line MG-63. We demonstrate that both TNF-alpha and TNF-beta dose- and time-dependently upregulate the mRNA levels of OPG. The effect is significant at and above 5 pM of TNF-alpha and 1 pM of TNF-beta. The stimulatory effect on OPG mRNA levels in MG-63 cells was detected after 2 hrs of incubation with TNF-alpha or TNF-beta. These data demonstrate that the expression of OPG in osteoblasts, with subsequent effects on osteoclastogenesis, is regulated by TNFs.

Viridans streptococci are a heterogeneous group of Gram-positive bacteria that are normal inhabitants of the mouth, upper gastrointestinal tract and oropharynx. These organisms are typically thought of as of low virulence, classically as the cause of infective endocarditis, although recently they have been implicated in serious infections in other settings. In particular, viridans group streptococci have been described as responsible for the alpha-streptococcal shock syndrome in neutropenic patients. The mechanism by which viridans streptococci cause bacteraemia associated with adult respiratory distress syndrome (ARDS) in these patients has not been elucidated. Using enzyme-linked immunosorbent assays, we compared the ability of cell-free bacterial supernatants derived from commensal and clinical strains of viridans streptococci to induce the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-alpha), tumour necrosis factor beta (TNF-beta) and interleukin 8 (IL-8) from human peripheral blood mononuclear cells (PBMC) in vitro. Supernatants of clinical isolates induced significantly more TNF-beta (P < 0.002) and IL-8 (P < 0.001) than did supernatants from commensal strains. The increased production of IL-8 by the clinical strains may be of importance in view of the role of IL-8 in the pathogenesis of the acute respiratory distress syndrome (ARDS), one of the principal clinical features of the alpha-streptococcal shock syndrome.

Mice with inactivated TNF-LT alpha genes have profound abnormalities of the immune system with hypoimmunoglobulinaemia, lack of lymph nodes, undifferentiated spleen, and defective Ig class switch. Transplantation of bone marrow cells from wild-type mice restored the synthesis of TNF, corrected the splenic microarchitecture, repopulated the lamina propria with IgA-producing plasma cells, and normalized the serum immunoglobulin levels of TNF-LT alpha deficient mice. Furthermore, the formation of germinal centers in the spleen and the defective Ig class switch in response to a T-cell-dependent antigen is corrected. These data demonstrate that most TNF-producing cells are bone-marrow-derived, and that the immunodeficiency due to TNF-LT alpha deletion can be corrected to a large extent by normal bone marrow, cell transplantation.

The potential role of tumour necrosis factors (TNFs) in autoimmunity and insulin-dependent diabetes mellitus (IDDM) led us to determine in vitro TNF-alpha and lymphotoxin-alpha (LT-alpha, TNF-beta) production in IDDM patients according to TNF polymorphism. LT-alpha production of peripheral blood mononuclear cells (PBMC) was lower in diabetic subjects (m = 0.30 +/- 0.2 ng.10(-6) cells) than controls (m = 0.68 +/- 0.3 ng.10(-6) cells, p < 0.05), and early age-at-onset was correlated with low LT-alpha production (rs = 0.8, p = 0.0006). TNF-alpha production was the same in patients and controls, but patients with HbA1c > or = 8% had a higher TNF-alpha production (m = 3.05 +/- 1.2 ng.10(-6) cells) than those with HbA1c < 8% (m = 1.31 +/- 0.33 ng.10(-6) cells, p < 0.05). A study of the microsatellite TNFa region close to the LTA gene showed that the presence of the TNFa1 allele in HLA-(DR3) subjects was associated with increased risk of IDDM. TNFa1-positive subjects (both patients and controls) also had lower LT-alpha production than other subjects. These results indicate that low LT-alpha production is an additional risk factor for IDDM and that poor glycaemic control in patients is associated with enhanced PBMC TNF-alpha production which causes an imbalance between TNF-alpha and LT-alpha production in IDDM patient.

The effects of prostaglandin E2 (PGE2) on lymphotoxin beta (LT-beta) and tumour necrosis factor alpha (TNF) were assessed in murine CD4+ Th1 and Th2 T cell clones. LT-beta mRNA was constitutively expressed by both T cell subsets. However, PGE2 inhibited its accumulation only in Th1, but not Th2 clones. PGE2 inhibited TNF mRNA accumulation and production and release of bioactive material by both Th1 and Th2 T cells. The effects of PGE2 were also evaluated on production of IL-3, another cytokine produced by both T cell subsets, and interleukin 4 (IL-4), which is produced only by Th2 cells. Though IL-3 was produced by both T cell subsets it was only inhibited in Th1 cells, a pattern similar to that observed for LT-beta. Accumulation of IL-4 mRNA in Th2 cells was not inhibited by PGE2. These results demonstrate that PGE2 does not affect LT-beta, IL-4, or IL-3 in Th2 cells, but inhibits TNF mRNA accumulation and production in this T cell subset. Thus, TNF appears to be the only cytokine susceptible to inhibition by PGE2 in Th2 cells. The fact that PGE2 inhibits LT-beta and IL-3 in Th1 but not Th2 cells points to a different mechanism of regulation of the same cytokine in different subsets. The mechanisms that contribute to TNF mRNA accumulation also may differ in the two CD4+ T cell subsets, because cycloheximide superinduced TNF mRNA in Th2 cells, but not in Th1 cells. The inhibitory effects of PGE2 on TNF mRNA accumulation by either T cell subset did not require de novo protein synthesis since preincubation with the protein synthesis inhibitor, cycloheximide, did not alter the PGE2-mediated effects. Cross-regulation of cytokine production and function has been demonstrated for both T cell subsets, and PGE2 may modulate the outcome of an immune response via differential regulation of cytokine production.

Endotoxin release from Pseudomonas aeruginosa treated with cell wall-active carbapenem antibiotics and its effect on the production of tumor necrosis factor alpha and nitric oxide were examined. Treatment of bacteria with imipenem induced much lower levels of endotoxin release than treatment with meropenem. The endotoxin released was demonstrated to be of the smooth type and O-specific polysaccharide-rich. The exposure of the filtrates of P. aeruginosa treated with imipenem to physiologically relevant cells caused low-level production of tumor necrosis factor alpha and nitric oxide, while similar treatment with meropenem induced high levels of production.

The interaction of LFA-1 on T lymphocytes with ICAM-1 on antigen presenting cells (APCs) is critical in determining conjugate formation between the APCs and T cells as well as activation of T cells. Recently, it was found that stimulation of THP-1 cells, a human monocyte M phi cell line, with Mycobacterium tuberculosis or its lipoarabinomannan, elicited the increase in the ICAM-1 expression. In addition, in cases of lepromatous leprosy patients with a serious defect in the M. leprae antigen-specific cellular immunity, keratinocytes in the leprosy lesions were lacking in the ICAM-1 expression. Therefore, ICAM-1 seems to participate in the host response to mycobacterial infections. Here, we studied the mode of the expression of ICAM-1 molecules on murine peritoneal M phis in response to stimulation with M. avium complex (MAC). In addition, the regulatory effect of some cytokines including TNF-alpha, IL-10, and transforming growth factor-beta (TGF-beta) on the ICAM-1 expression was studied. Monolayer cultures of peptone-starch induced murine peritoneal M phis were cultured in RPMI-1640 medium in the presence of MAC with or without test agents. At intervals, the M phis were stained with anti-ICAM-1 antibody (Ab) and then treated with alkaline phosphatase (Ap)-conjugated anti-1g Ab. After color development with NBT-BCIP substrate, percentage of the blue-stained (ICAM-1 positive) M phis was determined microscopically. The concentrations of TNF-alpha, IL-10, and TGF-beta in the M phi culture fluid was measured by ELISA using capture Ab, biotin-labelled capping Ab, and Ap-conjugated streptavidin. When M phis infected with MAC organisms were cultured in RPMI medium containing 10% fetal bovine serum or in serum-free GPI medium, a significant increase in their ICAM-1 expression was observed, reaching the peak at days 1 to 3, thereafter rapidly decreased and returned to the normal level by day 14. Further addition of TNF-alpha caused no significant change in the mode of the MAC-induced expression of ICAM-1. A transient increase in the IL-10 production of MAC-infected M phis was observed during the first 3-days cultivation, as in the case of TNF-alpha. On the other hand, TGF-beta production of the Mø population was initiated from day 3, and therafter increased gradually until day 14. ICAM-1 expression of the MAC-infected M phis was not influenced by the addition of IL-10, while anti-IL-10 Ab retarded the decline of ICAM-1 expression at day 7. On the other hand, the addition of TCF-beta attenuated the MAC infection-induced increase in the ICAM-1 expression significantly. In addition, anti-TGF-beta Ab significantly delayed the reduction of the ICAM-1 expression of MAC-infected M phis during days 3 to 7. These results indicate that, in the MAC-infected M phis. TGF-beta rather than IL-10 play important roles as a mediators for the late-phase down-regulation of ICAM-1 expression which had been transiently elevated by the action of TNF-alpha in the early phase of the Mø cultivation.

Background: The functional significance of cytokines expressed in situ by tumor cells and tumor infiltrating lymphocytes (TIL) in human colon carcinomas is largely unknown., Methods: We assessed TIL expression of interleukin-2 (IL-2), IL-4, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-10, and transforming growth factor-beta (TGF-beta), and tumor cell expression of IL-10 and TGF-beta in situ in 49 primary colon carcinomas and 20 metastases using immunohistochemistry., Results: The percentage of primary colon carcinoma samples in which > 20% of TIL expressed each cytokine was as follows: IL-4: 47%; TNF-alpha: 22%; TGF-beta: 10%; IFN-gamma: 6%; IL-2:2%; IL-10: 0%; and GM-CSF: 0%. Lymphocytes more commonly infiltrated colon carcinoma primaries than metastases, and TIL expression of IL-4 and TNF-alpha was more common in primary than metastastatic carcinomas. Expression of TNF-alpha by even a small proportion (> or = 3%) of the TIL in a colon carcinoma specimen was associated with better overall survival (P = 0.01) when compared with patients with little or no TIL TNF-alpha expression (5-year survival 82% vs. 47%). Expression of IL-4 by > or = 20% of colon carcinoma TIL was also associated with improved survival (P = 0.01; 5-year survival 87% vs. 50%). The expression of IL-10 or TGF-beta by colon carcinoma TIL or colon tumor cells themselves was not associated with impaired survival. Benign epithelial cells stained positively for IL-10 and TGF-beta more frequently than tumor cells (P < 0.001)., Conclusions: There are differences between the immune microenvironment of primary tumors and metastases. Although IL-10 is expressed by colon carcinoma cells and TIL, it is unlikely that it plays an important immunosuppressive role. TNF-alpha and IL-4 are commonly expressed by colon carcinoma TIL and both are associated with improved survival.

Burkitt's lymphoma (BL) represents a high malignant B cell tumour. It has been proposed that cytokines are responsible for some of the characteristics of BL. We have analysed a panel of different BL and lymphoblastoid cell lines (LCLs) for the expression of cytokines, including: IL 1 alpha, IL 1 beta, IL2, IL3, IL4, IL6, IL8, IL10, TNF alpha and TNF beta and for the soluble cytokine receptor for IL2 (slL2R). Our results show that expression of IL8, IL10, TNF alpha or TNF beta was detected frequently in several of the Burkitt or lymphoblastoid cell lines. There was a correlation between Epstein-Barr virus (EBV) infection and cytokine protein production. Our results suggest that EBV promote the expression of IL8, IL10, TNF alpha and TNF beta.

Study of the biosynthesis of human alpha 2-interferon and tumor necrosis factor in derivatives of E. coli SG200-50 recipient strain containing pIF16, pTNF311, and pLT21 plasmids demonstrated elimination of the plasmids from the cells without degradation thereof in the course of culturing. Methods increasing the content of the end products in the biomass are proposed.

The genes for tumour necrosis factor alpha (TNF alpha) and lymphotoxin alpha (LT alpha; TNF beta) are tandemly arranged in the central region of the MHC. They may, therefore, be of importance for the aetiology of MHC-associated diseases. The authors have prospectively studied the secretion of TNF alpha and LT alpha in relation to polymorphisms at positions -308 and -238 in the TNF alpha gene (TNFA), and two polymorphisms in the first intron of the LT alpha gene (LTA), as well as HLA-DR in 30 patients with chronic inflammatory bowel diseases (IBD) and 12 healthy controls. In the Dutch population, the alleles of these four polymorphisms are present in only five combinations, called TNF-haplotypes: TNF-C, -E, -H, -I, and -P. Significant associations between TNF haplotypes and TNF alpha and LT alpha secretion were found when PBMC were cultured with T-cell activators, irrespective of disease. Mean TNF alpha secretion of individuals carrying the HLA-DR3 associated TNF-E haplotype was significantly higher, as compared to individuals without this haplotype (26 441 pg/ml versus 19 629 pg/ml; P = 0.014). Individuals carrying the TNF-C haplotype produced the lowest amount of TNF alpha (17 408 pg/ml; P=0.022). The TNF-C and TNF-E haplotypes differ only at position -308 in the promoter of TNFA. Individuals carrying the HLA-DR1 associated TNF-I haplotype produced significantly less LT alpha when compared to those who lack this haplotype (1979 pg/ml versus 3462 pg/ml; P = 0.006). As the TNF-I haplotype is also associated with low TNF alpha secretion, this haplotype thus defines a 'low secretor phenotype'. In conclusion, this is the first study to show associations between TNF haplotypes and TNF alpha and LT alpha secretion when T-cell stimulators are used. These findings will contribute to define disease heterogeneity in IBD and may be of relevance for understanding the pathogenesis of autoimmune diseases.

Viridans streptococci (VS) have become recognized as an increasingly important cause of bacteraemia in neutropenic patients undergoing chemotherapy. Surprisingly, VS bacteraemia is associated with toxic shock-like syndrome (TSLS) and adult respiratory distress syndrome (ARDS), features not seen in non-neutropenic patients with viridans streptococcal bacteraemia. The mechanism by which these Gram-positive bacteria cause hypo-tension in the absence of endotoxin is not known. In this study, we have analysed the ability of cell-free bacterial supernatants derived from VS to induce the production of a number of cytokines from human peripheral blood mononuclear cells (PBMC). These cytokines were tumour necrosis factor alpha (TNF-alpha), tumour necrosis factor beta (TNF-beta) and interleukin 8 (IL-8). All 59 strains were able to induce these proinflammatory cytokines. We conclude that VS do produce secreted products which are able to stimulate the production of cytokines which may be important in the pathogenesis of shock caused by these bacteria.

Background: Mice with inactivated tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha) genes have profound abnormalities of the immune system including lymphocytosis, lack of lymph nodes, undifferentiated spleen, hypoimmunoglobulinaemia, and defective Ig class switch. Here, we asked whether this phenotype is due to incompetent lymphohemopoietic progenitors or to a defective environment., Materials and Methods: Lethally irradiated TNF-LT alpha-deficient and wild-type mice received bone marrow cells from either TNF-LT alpha-deficient or wild-type mice. The reconstitution and transfer of the phenotype was followed by morphological and functional analyses., Results: Bone marrow cells from wild-type mice restored the synthesis of TNF and LT alpha, corrected the splenic microarchitecture, normalized the lymphocyte counts in the circulation, and repopulated the lamina propria with IgA-producing plasma cells of TNF-LT alpha-deficient mice. Furthermore, the formation of germinal centers in the spleen and the defective Ig class switch in response to a T-cell dependent antigen was corrected, while no lymph nodes were formed. Conversely, the TNF-LT alpha phenotype could be transferred to wild-type mice by bone marrow transplantation after lethal irradiation., Conclusions: These data demonstrate that most TNF- and LT alpha-producing cells are bone marrow derived and radiosensitive, and that the immunodeficiency due to TNF-LT alpha deletion can be corrected to a large extent by normal bone marrow cell transplantation. The genotype of the donor bone marrow cells determines the functional and structural phenotype of the TNF-LT alpha-deficient adult murine host, with the exception of lymph node formation. These findings may have therapeutic implications for the restoration of genetically defined immunodeficiencies in humans.

The involvement of proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT) in multiple sclerosis is suggested by the parallel occurrence of these proinflammatory cytokines in acute and chronic active multiple sclerosis brain lesions. We describe the use of in situ hybridization with radiolabelled cDNA oligonucleotide probes to detect and enumerate TNF-alpha and LT mRNA expressing mononuclear cells without culture, and after culture in the presence of myelin basic protein (MBP), control antigens or without antigen. Compared with patients with aseptic meningo-encephalitis, non-inflammatory neurological diseases and healthy controls, the multiple sclerosis patients had elevated numbers of TNF-alpha and LT mRNA expressing mononuclear cells in blood when enumerated without previous culture, and also after culture with MBP. The MBP-induced upregulation of TNF-alpha and LT was major histocompatibility complex (MHC) class II molecule dependent. Tumour necrosis factor-alpha mRNA expressing mononuclear cells were further enriched in the multiple sclerosis patients' CSF. Positive correlations were observed in multiple sclerosis between TNF-alpha and LT mRNA expressing blood mononuclear cells, MBP-reactive TNF-alpha and LT mRNA expressing cells, and TNF-alpha and interferon-gamma (INF-gamma) mRNA expressing mononuclear cells. Upregulation of TNF-alpha correlated positively with exacerbation, enhanced disability and the secondary progressive phase of multiple sclerosis. Patients with optic neuritis, in many instances representing very early multiple sclerosis, had TNF-alpha and LT positive blood mononuclear cells that were elevated to the same extent as patients with clinically definite multiple sclerosis. The findings support the hypothesis that TNF-alpha and LT play a harmful role in the development of multiple sclerosis and suggest that TNF-alpha could be useful as a disease activity marker in multiple sclerosis.

Recent studies have shown that mucocutaneous leishmaniasis (MCL), a severe and debilitating form of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis infection, is accompanied by high circulating levels of tumor necrosis factor (TNF)-alpha. Analysis of TNF polymorphisms in Venezuelan ACL patients and endemic unaffected controls demonstrates a high relative risk (RR) of 7.5 (P < 0.001) of MCL disease in homozygotes for allele 2 of a polymorphism in intron 2 of the TNF-beta gene, especially in females (RR = 9.5; P < 0.001) compared with males (RR = 4; P < 0.05). A significantly higher frequency (P < 0.05) of allele 2 at the -308-basepair TNF-alpha gene polymorphism was also observed in MCL patients (0.18) compared with endemic control subjects (0.069), again associated with a high relative risk of disease (RR = 3.5; P < 0.05) even in the heterozygous condition. Because both the TNF-alpha and TNF-beta polymorphisms have previously been linked with functional differences in TNF-alpha levels, these data suggest that susceptibility to the mucocutaneous form of disease may be directly associated with regulatory polymorphisms affecting TNF-alpha production.

Lymphotoxin (LT) and tumor necrosis factor-alpha (TNF-alpha) play an important role in the pathogenesis of multiple sclerosis (MS). MS is associated with the HLA-DR2, Dw2, DQ6 HLA class II haplotype. Because both LT and TNF-alpha are encoded in the HLA region, the HLA association of MS may be related to the production of these cytokines. To test this hypothesis, we investigated the production of LT, TNF-alpha, and interferon-gamma (IFN-gamma) by CD4+ T-cell lines (TCLs) specific for myelin basic protein (MBP) or tetanus toxoid (TT) isolated from MS patients and normal controls. After stimulation with specific antigen but not mitogen, TCLs from HLA-DR2+ donors produced significantly more LT and TNF-alpha than TCLs from DR2- donors. In contrast, HLA-DR2+ and DR2- TCLs did not differ in the production of IFN-gamma, a cytokine also produced by T cells but not encoded in the HLA region. Increased secretion of LT and TNF-alpha was unrelated to the specificity (MBP vs TT), MHC restriction (HLA-DR2 vs other DR molecules), or source (MS vs normal) of the TCLs. There was no significant association of the cytokine production with individual LT or TNF-alpha alleles, indicating that the increased production of these cytokines may be linked to other polymorphic genes in this region. The results suggest that the association of MS with HLA-DR2 implies a genetically determined propensity of T cells to produce increased amounts of LT and TNF-alpha.

Background: Tumor necrosis factor alpha (TNF alpha) and beta (TNF beta) appear to play an important role in the regulation of the inflammatory response. The aim of the present study was to investigate the intrinsic capacity of peripheral blood mononuclear cells (PBMC) to produce these cytokines., Methods: PBMC from 41 patients with Crohn's disease (CD), with ulcerative colitis (UC), and 23 healthy controls (HC) were cultured for 48 h in the presence of the T-cell activators anti-CD3 and anti-CD28. Biologically active total TNF (TNF alpha and beta), TNF alpha, and TNF beta production were measured using a bioassay for biologically active TNF and specific TNF alpha and TNF beta enzyme-linked immunosorbent assays., Results: Large interindividual differences in TNF production were observed. Production of biologically active TNF after T-cell stimulation was significantly decreased in UC patients as compared with HC and CD patients (median, 337 U/ml, 800 U/ml, and 1050 U/ml, respectively). Stimulated TNF alpha production in UC patients (median, 432 U/ml) and in CD patients (median, 537 U/ml) did not differ statistically significantly from HC (median, 730 U/ml) as compared with HC and UC patients(median, 800 U/ml and 837 U/ml, respectively)., Conclusions: These findings support the concept that UC and CD are homogeneous, clearly distinguishable disease entities but rather a heterogeneous group of diseases. Studies directed to assess the immunogenetic background of these different disease manifestations in IBD are underway.

Tumor necrosis factor (TNF) production from T-cells stimulated with superantigenic exotoxins, staphylococcal enterotoxin B and streptococcal pyrogenic exotoxin A was investigated in the presence of cells bearing distinct isotypes of HLA class II molecules. The main T-cell subset for TNF production was investigated in parallel. Similarly high levels of TNF production were induced upon stimulation with the toxins in the presence of DR+ or DQ+ cells, but only marginal levels of TNF production were induced in the presence of DP+ cells. Although both CD4+ T-cells and CD8+ T-cells produced TNF-alpha and TNF-beta in response to toxin stimulation in the presence of HLA class II+ cells, the former T-cell subset was the major source of producers of TNF-alpha and TNF-beta.

One of the classical animal model systems in the study of important cardiovascular diseases, such as septic shock is that of swine. Important mediators of such disease states are different cytokines, among them tumor necrosis factor alpha (TNF-alpha) and beta (TNF-beta). This review aims to summarize the current knowledge about the primary structure and the regulation of the porcine TNF genes, as well as methods used to measure their respective proteins.

Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1 M) 2 to 3 wk after inoculation with CSA1 M cells produced IL-2, IFN-gamma, and TNF upon in vitro cultures. This was previously demonstrated to be a result of collaboration between tumor-primed CD4+ T cells and APCs binding CSA1 M tumor Ags in vivo. The IL-2- and IFN-gamma-producing capacities decreased with the progress of tumor-bearing stages. This was parallel to the levels of IL-2 and IFN-gamma mRNAs expressed by cultured spleen cells. In contrast, comparable levels of TNF mRNA were expressed by all groups of cultured cells. However, large amounts of TNF were secreted by the cells from early but not from late tumor-bearing mice. TNF was produced mainly by the non-T cell fraction upon stimulation with CD4+ T cell-derived IFN-gamma. Therefore, the reduced TNF production by whole spleen cells from late tumor-bearing mice was restored by addition of rIFN-gamma to their cultures. Reciprocally to the progressive decrease in the production of IFN-gamma/TNF, the capacities of tumor-bearing mice to produce TGF-beta and IL-6 increased along with tumor growth. TGF-beta suppressed production of IL-2, IFN-gamma, and TNF, but not of IL-6. Moreover, IFN-gamma/TNF production was negatively regulated by IL-6. Taken together with the fact that the growth of CSA1 M cells is completely inhibited by the combination of TNF and IFN-gamma, these results demonstrate that the tumor-bearing state induces an abnormal cytokine network under which the production of antitumor cytokines is negatively regulated.

A critical event in B cell immortalization by Epstein-Barr virus (EBV) is the establishment of an autocrine loop where cells produce a growth factor which supports their own proliferation. We investigated the potential of lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) cell lines to produce and respond to the cytotoxins, tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT). Transformation in vitro of peripheral blood B cells by EBV from seven different donors resulted in spontaneous production of both LT (11,542 pg/ml +/- 7546, mean +/- SD) and, to a lesser extent, TNF-alpha (197 pg/ml +/- 174). Similarly BL cell lines derived from in vivo transformation which developed a 'LCL-like' phenotype in vitro (group III) produced more LT (1990 pg/ml +/- 1740) than the 'group I' BL cell lines (< 40 pg/ml LT) which had maintained the original BL biopsy cell phenotype in vitro. Transformation of peripheral blood B cells to generate LCL also resulted in an increase in surface p75 (p < 0.02) and to a lesser extent p55 (not significant, ns) TNF receptor (TNF-R) expression. Similar increases in surface TNF-R (p75 p < 0.02, p55 ns) were observed on the 'group III' BL cell lines compared with the 'group I' BL cell lines. Proliferation of an LCL and a 'group III' BL cell line in vitro was via an autocrine loop since inhibition of LT reduced proliferation. This proliferation could also be blocked in the presence of the antagonistic anti-p55 TNF-R antibody, H398, but not the antagonistic antibody anti-p75 TNF-R antibody UTR-1. Furthermore, proliferation could be induced with the p55 agonistic antibody, HTR-9. In contrast to these observations with p55 TNF-R antibodies, two out of six of the 'group III' BL lines (Jijoye and Oba) only expressed the p75 TNF-R and proliferation of these cells could only be blocked by the antagonistic anti-p75 TNF-R antibody UTR-1. These data suggest that LT is an autocrine growth factor for lymphoblastoid cells, and BL cell lines which display an LCL phenotype. Furthermore, although both TNF-R are increased on the surface of these cells, this autocrine growth signal is mediated principally through binding to the p55 TNF-R.

Based on the premise that naturally occurring glycosaminoglycans could serve as building blocks for synthesizing nontoxic drugs for suppression of tumor necrosis factor (TNF) production by inflammatory cells, we have chemically modified hyaluronic acid (HA) and tested its effects in blocking TNF-alpha and TNF-beta production in vitro. HA was chosen mainly for its structural simplicity, nonimmunogenicity, and readiness for chemical modifications. When HA was chemically polysulfated to a sulfate/hexosamine molar ratio of 3.9, the sulfated HAs was shown to be a potent inhibitor of TNF-alpha production in lipopolysaccharide (LPS)- or interferon-gamma-activated THP-1 cells. For example, a concentration of HAs as low as 10 ng/ml reduced TNF-alpha production in LPS-activated THP-1 cells more than 50%, whereas achieving a similar extent of reduction required 50 micrograms/ml native HA. By decreasing the extent of polysulfation, the inhibitory effect of HAs on TNF-alpha production was diminished. Other chemical modifications, including deacetylation, thiolation, or reduction of the carboxylic groups, could not increase the efficacy of HA in suppression of TNF-alpha production. Naturally polysulfated glycosaminoglycans, such as chondroitin sulfates, keratan sulfate, heparan sulfate, and heparin, failed to inhibit TNF-alpha production. HAs also restricted TNF-beta (lymphotoxin) secretion in an Epstein-Barr virus-transformed B cell line, Roha-9, which constitutively produces TNF-beta. HAs had no inhibitory effect on the proliferation of THP-1 or Roha-9 cells, which would account for the reduced TNF-alpha or TNF-beta production. Furthermore, time-course metabolic labeling studies revealed that HAs could not restrict overall protein synthesis and secretion in THP-1 cells. However, HAs increased complement C1q secretion in THP-1 in a dose-dependent manner, but it had no effect on biosynthesis of complement C1 inhibitor, factor D, and Fc gamma receptor type II (Fc gamma RII). These results indicate that HA, selectively restricts the production of TNF-alpha, TNF-beta, and probably several other protein species.

The capacity of human T cell subsets, CD4+ or CD8+ T cells, to produce tumour necrosis factors (TNF-alpha and TNF-beta) upon stimulation with toxic shock syndrome toxin-1 (TSST-1) and the requirement for MHC class II molecules on accessory cells (AC) in the response were investigated. The capacity of CD4+ T cells was much higher than that of CD8+ T cells in TSST-1-induced production of TNF-alpha and TNF-beta. The expression of MHC class II molecules on AC was required in the response.

Experimental infection of BALB/c mice with Leishmania major leads to lesions which progress without healing and visceralization, reproducing the most severe forms of human leishmaniasis, while resistant mice like CBA spontaneously resolve lesions and develop protective immunity. Given the conflicting data pertaining to the role of tumor necrosis factor alpha (TNF) in Leishmania infection, we analyzed the expression of TNF, tumor necrosis factor beta (lymphotoxin), and TNF receptor type I (TNF-RI) and type II (TNF-RII) genes in vivo and correlated TNF gene expression in vivo with the production of biologically active TNF by lymphoid cells in vitro. No significant difference in the expression of TNF mRNA was found between susceptible and resistant strains of mice during the course of infection. The depletion of CD4+ T cells in vitro did not change the level of TNF mRNA in BALB/c lymph node cells but led to the total disappearance of TNF mRNA in CBA mice. Unprimed spleen cells did not produce detectable amounts of TNF, whereas 1 week after infection, TNF bioactivity was detected and increased in both strains of mice until 5 weeks of infection. While neutralization of TNF activity in vivo did not alter the course of infection in BALB/c mice, in CBA mice it led to an increase in lesion size and a delay in the healing process but did not interfere significantly with the outcome of infection. Finally, no significant difference in the levels of lymphotoxin, TNF-RI, or TNF RII mRNA expression was found between both strains. The information resulting from these investigations supports the notion that, in vivo, TNF is not the decisive factor responsible for the resistant versus susceptible phenotype in leishmania infection.

The present study shows that the decrease of TNF alpha in postburn hypertrophic scars is due to a decrease in the steady-state level of TNF alpha mRNA and thus to an altered biosynthesis of the cytokine. Thirteen scars, including seven hypertrophic and six normotrophic scars, were tested for TNF alpha mRNA production by a semiquantitative reverse polymerase chain reaction (PCR) method. TNF beta and beta actin were tested as a control. Six out of six normotrophic scar samples amplified with primers for TNF alpha showed a positive PCR signal up to the 1:32 dilution. On the contrary all the hypertrophic tested samples (7/7) had a positive PCR signal only at the 1:1 or 1:2 dilution. All samples, both normotrophic and hypertrophic, were homogeneous as to TNF beta production.

Expression of cytokine genes, TNF-alpha, TNF-beta and IFN-gamma, in human astroglial cell lines and in fresh brain specimens was studied by PCR. mRNA transcripts of TNF-alpha could be detected in three out of five astrocytomas and neuroblastoma cell lines, and after stimulation with IL-1 beta/IFN-gamma or LPS/IFN-gamma all these cell lines expressed TNF-alpha genes. TNF-beta genes could not be detected in these cell lines. We were able to detect expression of IFN-gamma genes within two astrocytoma cell lines, which interestingly did not show TNF-alpha activity. In addition to the cultured cells, we also examined gene expression of these cytokines within four human malignant astrocytoma specimens, two peritumoral brain and two autopsied normal brains. The results show that tumour and surrounding reactive lesions express TNF-alpha genes (four of six) but not normal brains. The concentration of these cytokines in the supernatant of cultured cells was measured quantitatively by TNF-alpha, -beta or IFN-gamma ELISA. The combined stimulation of these neuroglial cell lines with IL-1 beta and LPS or IFN-gamma, revealed a high level of TNF-alpha activity. This was especially evident with a neuroblastoma cell line. The concentration of TNF-alpha in the supernatant of the IMR32 neuroblastoma cell line increased markedly upon stimulation with IL-1 beta in both a time- and dose-dependent fashion in the presence of LPS or IFN-gamma. Next, we examined expression of IL-1 beta and IFN-gamma genes in the brain specimens. The result shows that four in six tumour and peritumoral regions expressed IFN-gamma genes and one specimen showed IL-beta gene by PCR. From these experiments it is suspected that neuroglial cell-derived TNF-alpha induced by IL-1 beta of IFN-gamma may participate in local immune reactions of the brain in an autocrine and paracrine fashion.

Tumor necrosis factor (TNF) and lymphotoxin (LT; TNF-beta) are major cytokines produced by B lymphocytes. Stimulation by okadaic acid, a phosphatase 1 and 2A inhibitor, markedly increased TNF mRNA accumulation and cytokine production. On the other hand, the accumulation of LT mRNA was not affected by okadaic acid despite structural and functional similarities between TNF and LT. The increase in TNF mRNA accumulation was due to the stimulation of gene transcription and a marked stabilization of this mRNA. The binding activities of the transcription factors AP-1 and AP-2 and NF kappa B, which regulates TNF gene transcription, were also stimulated by okadaic acid. In addition, okadaic acid was shown to increase TNF production at the protein level. These results show the importance of protein phosphatases in the regulation of cytokine production in B cells, and further identifies differences in the regulation of TNF-alpha and LT production.

The expression of the cytotoxic cytokines tumor necrosis factor alpha and TNF beta or lymphotoxin (LT) was assessed in rat cardiac transplants during rejection. Newborn rat cardiac grafts placed in adult rat ear pinnae were retrieved on days 1 through 10 posttransplantation; the average time to rejection, assessed by the absence of detectable electrocardiographic activity, was determined to be 7 days. Total cellular RNA and tissue homogenates were prepared from cardiac transplants in order that relative levels of TNF alpha and LT mRNA and TNF protein could be determined. A biphasic pattern of TNF alpha gene expression was consistently seen in cardiac allografts. TNF alpha mRNA transcripts were detected as early as day 2 post-tx, with peak levels appearing on day 3 post-tx. Although transcript levels decreased by day 4, a significant increase appeared again on day 6 post-tx, coincident with the onset of rejection. Similar to TNF alpha gene expression, LT transcripts demonstrated a biphasic pattern of induction. LT mRNA transcripts also reached peak levels on day 3 post-tx, with a second increase in transcript levels coincident with rejection. TNF protein levels in allografts displayed a biphasic pattern, similar to that shown by the cytokine mRNAs. Peak levels of TNF protein were detected on day 3 post-tx, with a second increase again coinciding with rejection. In contrast to TNF expression found in allografts, TNF alpha and LT mRNA transcripts were not detected in isografts on days 1 through 10 post-tx. TNF protein levels in cardiac isografts were consistently at or below the standard limits of detection, and on days 3 through 7 post-tx were significantly reduced (P < or = 0.001) when compared with time-matched allografts. Increased expression of the cytotoxic cytokines TNF alpha and LT, therefore, appears to be allograft-specific and is an early event during rat cardiac allograft rejection. In conclusion, induction of TNF gene expression may be an important early indicator of transplant rejection.

The screening of a bovine genomic library with a human tumour necrosis factor-alpha (TNF-alpha) cDNA probe resulted in the isolation of a 7.2 kb DNA fragment containing the entire bovine TNF-alpha gene. Analysis of this genomic clone showed that it also contains the bovine lymphotoxin (LT, TNF-beta) gene. Comparison to published sequences of human, murine, ovine and rabbit counterparts allowed us to delineate the coding sequences, the promoters and the enhancers of these two genes. Sequences involved in the regulation of translation and in the mRNA stability were found in the 3' untranslated regions.

In vitro effects of human recombinant IL-6 (1-1000 U/ml) on highly enriched human NK CD3-CD56+ cells (94% +/- 2; mean +/- SEM; n = 8), obtained from PBL were studied. IL-6 induced low levels of NK cell proliferation (7- to 30-fold during 6-day incubation), which was IL-2-independent, because IL-6 did not induce detectable IL-2 production by NK cells. Two-color flow cytometry analysis demonstrated that incubation of NK cells with IL-6 at the optimal concentration of 250 U/ml for 6 days significantly increased the proportion of NK cells expressing the following activation Ag: CD25 (26% +/- 17, mean +/- SEM vs 4% +/- 1 in control, n = 5), CD54 (44% +/- 17 vs 9% +/- 3), HLA-DR (29% +/- 13 vs 12% +/- 4), CD69 (45% +/- 7 vs 12% +/- 3), and CD71 (34% +/- 17 vs 6% +/- 2). The mean fluorescence intensity of these activation Ag was increased as well. IL-6 induced expression of CD49b (alpha-chain of VLA-2, 20% +/- 11 vs 2% +/- 1) and CD49c (alpha-chain of VLA-3, 43% +/- 17 vs 5% +/- 3), which are not expressed on resting NK cells. IL-6 also enhanced the fluorescence intensity of beta 1 integrins, CD49d, CD49e, and CD49f, expressed on NK cells. IL-6-stimulated NK cells showed significantly increased integrin-mediated adhesion to fibronectin- or laminin-coated plates (26 +/- 3 mean % cells adhering +/- SEM vs 15 +/- 4 in control for FN and 19 +/- 1 vs 11 +/- 1 for LM, p < 0.05 for both) as determined in a 3 h binding assay. As assessed by inhibition of adhesion using mAb to the VLA-2, -3, -4, -5, and -6, NK cell adhesion to fibronectin was mediated by VLA-4 and 5, and their adhesion to laminin by VLA-3 and -6. NK cells incubated in the presence of IL-6 were found to produce a factor cytostatic to WEHI-164 clone 13 target cells. This effect was partly, although significantly, blocked by neutralizing antibodies to TNF-alpha or TNF-beta. Our data demonstrate that IL-6 can directly activate human NK cells, but is a less potent NK cell activator, for all activation and functional parameters studied, than IL-2.

To understand the role of TNF in the regulation of inflammation and the development of autoimmune diseases such as insulin-dependent diabetes mellitus, we produced transgenic mice in which the synthesis of murine TNF-alpha was directed by the rat insulin II promoter. The expression of the TNF-alpha transgene was restricted to the pancreas, in contrast to TNF-beta expression from the same promoter, in which the transgene was expressed in the pancreas, kidney, and skin. The expression of TNF-alpha in the pancreas of transgenic mice resulted in an overwhelming insulitis, composed of CD4+ and CD8+ T cells and B220+ B cells, considerably greater than that of TNF-beta transgenics. Moreover, in contrast to the predominant peri-insulitis observed in TNF-beta transgenic mice, the majority of the infiltrate in the TNF-alpha transgenic mice was within the islet itself. These unique patterns of infiltration were observed in the F1 progeny of crosses with C57BL/6 as well as NOD. Both TNF-alpha and TNF-beta transgenic mice show elevated expression of leukocyte adhesion molecules VCAM-1 and ICAM-1 in islet endothelia and increased expression of MHC class I on islet cells. This inflammation did not result in reduced insulin content of the islets, nor did it lead to diabetes. These data suggest that additional stimuli are necessary to initiate the process of islet destruction.

Murine embryo fibroblasts (MEF) were found to secrete tumor necrosis factor (TNF) in response to stimulation with endotoxin. Endotoxin-induced TNF production by MEF was inhibited by cycloheximide. However, reversal of the effect of this inhibitor on protein synthesis results in TNF being secreted in amounts equivalent to those produced by endotoxin-induced MEF not treated with cycloheximide. Actinomycin D treatment of MEF blocked the production of endotoxin-induced TNF. Maximal production of TNF required MEF gene transcription during the first 6 h of incubation with endotoxin. To determine whether endotoxin-induced TNF alpha (TNF-alpha) and/or TNF beta were produced by MEF, cDNA was synthesized from the total RNA isolated from endotoxin-induced MEF and amplified by the polymerase chain reaction in the presence of oligonucleotide primers specific for each cytokine. On the basis of the polymerase chain reaction analysis, it was determined that TNF-alpha mRNA levels were increased in endotoxin-induced MEF. Thus, production of TNF-alpha by fibroblasts in response to the endotoxin component of bacterial cell walls is likely to contribute to the expression of TNF-mediated effects occurring in fibroblast-rich tissues infected with gram-negative bacteria.

We have previously reported the isolation of the cytotoxic factors, tumor necrosis factor-alpha (TNF-alpha), from monocytes and lymphotoxin or TNF-beta, from an established human B lymphoblastoid cell line. In the current study, we examined the production of cytotoxic factors by primary lymphoid cells derived from patients with different malignancies and correlated it with the chromosomal abnormalities in producer cells. Lymphoid cell lines were established from 78 untreated patients with breast carcinoma, 2 with cervical carcinoma, 24 with colon carcinoma, 12 with prostate carcinoma, 3 with melanoma, and the remaining from asymptomatic family members. Lymphoid cells from all 119 patients and their asymptomatic family members constitutively produced as much as 240 units/ml of TNF-beta in vitro. In contrast, TNF-alpha was produced by lymphoid cells from only 6 patients; a related cytokine with similar biological properties to TNF-beta. Sixty-five of these 119 samples were also analyzed for chromosomal abnormalities by standard cytogenetic technique. The production of TNFs was accompanied with varying degrees of chromosomal abnormalities in the cells from all patients. These included both structural and numerical abnormalities. We also examined the direct effect of TNF-beta on the induction of chromosomal abnormalities in growing cultures of both T and B lymphocytes. Our preliminary results indicate that treatment of lymphocytes with this cytokine induced chromosomal rearrangements in a dose-dependent manner. Thus, we provide for the first time both indirect and direct evidence that a soluble mediator such as TNF-beta can induce chromosomal alterations commonly associated with different types of tumors.