Human T lymphotropic virus I infection deregulates surface expression of the transferrin receptor

Human T-lymphotropic virus I (HTLV-I) is an etiologic agent in adult T cell leukemia. In an effort to understand the relationship between HTLV-I infection and malignant transformation, we have examined transferrin receptor expression in HTLV-I-infected cells. Transferrin receptor expression in normal T cells is tightly regulated and essential for cell proliferation. We have used matched T cell sets originating from a normal donor, consisting of tetanus toxoid-specific normal T cell clones (TM3 and TM5) and their in vitro HTLV-I-infected counterparts (TM3H and TM5H). Using these matched sets of virus-infected and normal T cells, we have determined that HTLV-I infection leads to hyperexpression of surface transferrin receptors (five- to six-fold higher than normal counterparts). Although the growth rates of the virus-infected cells did not differ significantly from their normal controls, HTLV-I-infected cells constitutively hyperexpressed surface transferrin receptors, whereas the level of surface receptor expression of normal counterpart cells varied during the cycle of antigenic stimulation. Immunoprecipitation of total (surface plus cytoplasmic) transferrin expression showed that the HTLV-I-infected cells did not possess a greater total number of transferrin receptors than their normal counterparts. This data was supported by Northern blot analysis, which showed equivalent transferrin receptor mRNA expression in HTLV-I-infected and uninfected cells. Functional analysis revealed a marked defect in 59Fe-transferrin internalization in the HTLV-I-infected cells. Furthermore, the HTLV-I-infected cells showed markedly decreased transferrin receptor phosphorylation and internalization in response to active phorbol ester. Thus the data demonstrate that in peripheral blood T cells, HTLV-I infection is accompanied by surface transferrin receptor overexpression secondary to subcellular redistribution and defective internalization.     

Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.     

Differential expression of IL-1 and TNF receptors in murine macrophages infected with virulent vs. avirulent Legionella pneumophila

Infection of macrophages from genetically susceptible A/J mice with Legionella pneumophila induces high levels of various cytokines in serum as well as in cultures of spleen or peritoneal cells from the mice. However, modulation of receptor expression for these cytokines during infection has not been studied in detail, even though these receptors on macrophages have a critical role in inflammatory responses during the infection. In the present study, the differential expression of mRNA for TNF and IL-1 receptors as well as receptor antigens during infection of macrophages with virulent vs. avirulent L. pneumophila was investigated. Mouse thioglycollate-elicited peritoneal macrophages showed by RT-PCR constitutive steady-state levels of mRNA for TNF-type I and -type II receptors as well as IL-1 type I receptor. However, IL-1 type II receptor mRNA was not expressed in thioglycollate-elicited macrophages. Infection of macrophages with virulent bacteria caused an upregulation of IL-1 type I and TNF type I receptor mRNA, but had no effect on TNF type II receptor message. Avirulent L. pneumophila infection caused much less induction of these receptor mRNAs. The amount of receptor antigen of IL-1 type I on the surface of macrophages was also increased by infection with virulent L. pneumophila determined by flow cytometric analysis. These results indicate that L. pneumophila infection not only causes induction of various cytokines, but also modulation of certain cytokine receptors, which may regulate the susceptibility to infection.     

Differential effect of IL-18 on endothelial cell apoptosis mediated by TNF-alpha and Fas (CD95)

Interleukin-18 (IL-18) is a newly identified cytokine with proinflammatory activity. Numerous studies have shown that proinflammatory cytokines may regulate endothelial cells (EC) apoptosis mediated by members of the tumor necrosis factor (TNF) family, such as TNF-alpha and Fas. In this study we hypothesized that IL-18 may regulate the susceptibility of liver endothelial cells (LEC) to apoptosis induced by TNF and Fas. IL-18 increased the susceptibility of LEC to undergo apoptosis mediated by TNF but not by Fas. Since TNF-induced apoptosis is mediated by the type I TNF receptor (TNFRI), we investigated up-regulation of this receptor in IL-18-treated LEC. IL-18 induced up-regulation of the TNFRI on the surface of LEC. Partial blocking of LEC apoptosis induced by IL-18 and TNF was observed when the cells were pretreated with the broad-spectrum inhibitor of caspases z-VAD-fmk, suggesting involvement of the caspase pathway in apoptosis induced by these cytokines in these cells. Our results show that IL-18 differentially regulates apoptosis mediated by the death-inducing factors, TNF and Fas. To our knowledge, this is the first report that IL-18 may regulate endothelial cell apoptosis mediated by TNF. These results may have clinical implications in those clinical hepatic conditions associated with high levels of IL-18 and TNF.     

Tumor necrosis factor alpha and interferon gamma modulate the expression of the vitronectin receptor (integrin beta 3) in human endothelial cells.

In this paper we show that tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) alter the expression of extracellular matrix receptors (integrins) in cultured human endothelial cells. Endothelial cells express at their surface integrins of the beta 1 and beta 3 groups that include receptors for fibronectin, collagen, laminin, and vitronectin. After treatment for 72 h with a combination of TNF alpha and IFN gamma, the level of the vitronectin receptor (alpha v beta 3) at the cell surface decreases by 70%, whereas the amounts of the beta 1 integrins remain unchanged. The decreased expression of the alpha v beta 3 complex at the cell surface is due to a selective effect of TNF alpha and IFN gamma on the regulation of the beta 3 subunit synthesis at the translational level. In fact, although the steady state levels of the mRNA for the beta 3 subunit are comparable in control and treated cells, the overall synthesis of the beta 3 subunit is decreased by a factor of 70%. No significant alteration of the synthesis of the companion alpha v subunit is detectable in cytokine-treated cells. As a consequence of the decreased expression of the receptor, cytokine-treated cells show decreased ability to adhere to vitronectin but adhere normally to fibronectin. These data show that two important inflammatory mediators, TNF alpha and IFN gamma, can modify the interaction of endothelial cells with the extracellular matrix by selectively altering the expression of specific cell surface integrin complexes.     

Ligand-independent down-regulation of IFN-gamma receptor 1 following TCR engagement

Activated T lymphocytes modulate the level of many molecules on their cell surface, including cytokine receptors. This regulation of cytokine receptor expression affects the ability of T cells to respond to cytokines and thus influences the outcome of an immune response. The receptor for IFN-gamma, a proinflammatory cytokine, consists of two copies of a ligand binding chain (IFN-gammaR1) as well as two copies of a second chain (IFN-gammaR2) required for signal transduction. The expression of IFN-gammaR2 is down-regulated at the mRNA level on CD4+ T cells when they differentiate into the Th1, but not the Th2, phenotype. This down-regulation has been demonstrated to depend on the ligand, IFN-gamma, which is produced by Th1 but not Th2 T cells. The regulation of the cell-surface expression of IFN-gamma receptors during primary T cell activation has not been reported. Naive and differentiated T lymphocytes express IFN-gammaR1 at the mRNA level and as a cell-surface protein. In this study, we present evidence that cell-surface expression of IFN-gammaR1 is transiently down-regulated on the surface of naive CD4+ T cells shortly after TCR engagement. Furthermore, this down-regulation is not mediated by the ligand, IFN-gamma, but results from TCR engagement and can be inhibited by cyclosporin A.     

Etk/Bmx as a tumor necrosis factor receptor type 2-specific kinase: role in endothelial cell migration and angiogenesis

Tumor necrosis factor (TNF) is a cytokine that mediates many pathophysiologial processes, including angiogenesis. However, the molecular signaling involved in TNF-induced angiogenesis has not been determined. In this study, we examined the role of Etk/Bmx, an endothelial/epithelial tyrosine kinase involved in cell adhesion, migration, and survival in TNF-induced angiogenesis. We show that TNF activates Etk specifically through TNF receptor type 2 (TNFR2) as demonstrated by studies using a specific agonist to TNFR2 and TNFR2-deficient cells. Etk forms a preexisting complex with TNFR2 in a ligand-independent manner, and the association is through multiple domains (pleckstrin homology domain, TEC homology domain, and SH2 domain) of Etk and the C-terminal domain of TNFR2. The C-terminal 16-amino-acid residues of TNFR2 are critical for Etk association and activation, and this Etk-binding and activating motif in TNFR2 is not overlapped with the TNFR-associated factor type 2 (TRAF2)-binding sequence. Thus, TRAF2 is not involved in TNF-induced Etk activation, suggesting a novel mechanism for Etk activation by cytokine receptors. Moreover, a constitutively active form of Etk enhanced, whereas a dominant-negative Etk blocked, TNF-induced endothelial cell migration and tube formation. While most TNF actions have been attributed to TNFR1, our studies demonstrate that Etk is a TNFR2-specific kinase involved in TNF-induced angiogenic events.     

Tumor necrosis factor modulates epidermal growth factor receptor phosphorylation and kinase activity in human tumor cells. Correlation with cytotoxicity

Tumor necrosis factor (TNF) is a cytokine which induces cytotoxicity in some but not all tumor cells. Initial studies of five tumor cell lines demonstrated that TNF was able to rapidly (within 30 min) modulate tyrosine protein kinase activity of epidermal growth factor (EGF) receptors on tumor cell lines which were sensitive to the cytotoxic effects of TNF but not alter EGF receptor kinase activity in TNF-resistant tumor cells. Two tumor cell lines (ME-180 cervical carcinoma and T24 bladder carcinoma) which have been shown to express similar TNF-binding characteristics but differ in their sensitivity to the cytotoxic actions of TNF were chosen for further characterization. Treatment of TNF-sensitive ME-180 cells with 1 nM TNF resulted in a 3-fold stimulation of EGF receptor tyrosine protein kinase activity within 10 min which correlated with increased phosphorylation of EGF receptor protein itself. In addition, dose-response studies indicate that similar concentrations of TNF modulate both ME-180 cell growth and EGF receptor kinase activity. Treatment of TNF-resistant T24 cells showed that TNF had no significant effect on their growth, EGF receptor tyrosine protein kinase activity, or phosphorylation of EGF receptor protein although EGF receptor kinase activity was stimulated by EGF. Quantitation of receptors expressed on the surface of ME-180 and T24 cells demonstrated a 3-fold difference between the number of EGF-binding sites on T24 (100,000) versus ME-180 cells (300,000), suggesting the relative abundance of EGF receptor does not solely account for differential effects of TNF on EGF receptor activation in these two cell lines. Phosphoamino acid analysis of EGF receptor from 32P-equilibrated ME-180 cells demonstrated that TNF-induced phosphorylation of amino acids which was quantitatively similar to that of EGF but distinct from the effects of phorbol ester. However, unlike EGF, TNF was unable to stimulate EGF receptor kinase activity in ME-180 cell lysates. The kinetics of EGF receptor activation and the metabolic consequence of activation of EGF receptor activity by TNF appear to be distinct from those induced by EGF. These results suggest that TNF-induced modulation of EGF receptor occurs through a unique mechanism and may play a role in the cytotoxic actions of TNF.     

Tumour necrosis factor receptor I (p55) is upregulated on endothelial cells by exposure to the tumour-derived cytokine endothelial monocyte- activating polypeptide II (EMAP-II)

Endothelial monocyte activating polypeptide-II (EMAP-II) is an inflammatory cytokine known to have a role in neutrophil and macrophage chemotaxis and in apoptosis. It is a tumour-derived cytokine that sensitizes tumour vasculature to the effects of systemic TNF. In order to gain insight into the mechanism by which EMAP-II sensitizes vessels to TNF, we focused on its effects on TNF receptor expression. In human umbilical vein endothelial cells (HUVEC), TNF-R1 mRNA is increased four-fold following incubation with recombinant EMAP-II. Conditioned media from cell lines known to produce high levels of EMAP-II upregulated TNF-R1 but not TNF-R2 by up to twenty-fold compared to media controls and low expressing cell lines; this effect was blocked by anti-EMAP-II antibody. Recombinant EMAP-II upregulated TNF-R1 expression by approximately six-fold. Analysis of HUVEC lysates by ELISA showed increased expression of TNF-R1 within 2 h; TNF-R2 expression was unaffected by recombinant EMAP-II. Finally, immunohistochemistry of human melanomas in vivo showed that TNF-R1 staining is increased on the vessels of tumours known to express high levels of EMAP-II compared to low EMAP-II expressing tumours. These results suggest that EMAP-II upregulates TNF-R1 expression by endothelial cells both in vitro and in vivo. This induction of TNF-R1 expression may be the mechanism by which EMAP-II sensitizes tumour endothelium to the effects of TNF leading to haemorrhagic necrosis.     

Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells.

Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.     

Enhancement of IL-6 receptor beta chain (gp130) expression by IL-6, IL-1 and TNF in human epithelial cells.

Analysis of the IL-6 Receptor beta chain (gp130) mRNA expression on the two human epithelial cell lines UAC and Hep3B reveals that it is enhanced by IL-6, IL-1 and TNF treatment. In the case of UAC cells, TNF action might be mediated by IL-6. For Hep3B cells, TNF seems to exert a direct effect on gp130, as no IL-6 expression is detected after stimulation by this cytokine. On the same cells, increase of the binding of an anti-gp130 monoclonal antibody was observed after treatment by TNF, which denotes the effective appearance of new gp130 molecules on the cell surface. All this cytokines seem to act selectively on the beta chain of the IL-6 receptor. This probably reflects the importance for some cells to have gp130 represented on their membrane in inflammatory contexts.     

Macrophage-neutrophil interaction: a paradigm for chronic inflammation revisited

Macrophages have been described as 'factories' of pro-inflammatory cytokines. Several years ago the present investigators reported that binding of inactive myeloperoxidase (iMPO) to the macrophage-mannose receptor resulted in the induction of TNF and other cytokines. Also, if endothelial cells were incubated with iMPO, but not enzymatically active myeloperoxidase (MPO), upregulation of cytokine mRNA and cytokines was observed. Taken in their entirety, the data suggest a dichotomy of function for myeloperoxidase; that is, enzymatically active MPO functions primarily in cell killing through the 'cytotoxic triad' and iMPO functions as an immunoregulatory molecule through the induction of numerous cytokines. These studies underscore a previously unrecognized interaction among neutrophils, endothelial cells and macrophages, resulting in the induction of TNF and perpetuation of inflammation. The inflammation induced could be relevant in a number of diseases in which neutrophils play a prominent role. The importance of this interaction in the pathogenesis of rheumatoid arthritis is currently under investigation.     

Up-regulation of thrombomodulin in human umbilical vein endothelial cells in vitro

Previous studies have shown that thrombomodulin (TM) on endothelial cells is down-regulated by endotoxin, interleukin-1 beta (IL-1 beta), and tumor necrosis factor (TNF). This loss of anti-coagulant potential is thought to be related to the hypercoagulable state in sepsis, inflammation, and cancer. The current studies describe up-regulation of TM in human umbilical vein endothelial cells (HUVECs) by several compounds as judged by increased surface cofactor activity, surface TM antigen, and TM mRNA levels. Surface TM activity was increased by active phorbol esters (10(-8) M, 24-48 h), analogs of cAMP (1-10 mM, 4 h), and forskolin (10(-5) M, 24-48 h). Up-regulation of TM in HUVECs by 4 beta-phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (dBcAMP) was due to de novo synthesis of TM protein resulting from increased TM mRNA levels. The results suggest that protein kinase C and protein kinase A may be involved in cellular regulatory mechanisms for TM expression. In addition, PMA effects on surface TM activity are biphasic, with an initial reduction followed by a significant enhancement. Hence, we propose that compounds capable of increasing intracellular cAMP concentrations in HUVECs may be useful in preventing thrombosis by increasing the anti-thrombotic properties of endothelial cells.     

Hypoxia enhances CXCR4 expression in human microvascular endothelial cells and human melanoma cells

The influence of environmental factors (cytokines, matrix components, serum factors and O(2) level) on expression of receptors for angiogenic versus angiostatic CXC chemokines in human microvascular endothelial cells has not been extensively investigated. Our semi-quantitative RT-PCR analysis demonstrated that TNF-alpha and IFN-gamma repressed CXCR4 mRNA levels in immortalized human microvascular endothelial HMEC-1 cells after 4 h, whereas only TNF-alpha displayed inhibitory activity in primary human microvascular endothelial cells (HMVEC). CXCR4 mRNA expression was not affected by VEGF, GM-CSF, IL-1beta or various basal membrane matrix components, but was significantly up-regulated after serum starvation and/or hypoxic treatment of the microvascular endothelial cells. The alternative CXCL12 receptor, CXCR7/RDC1, was also up-regulated by hypoxia in HMEC-1 cells, although less consistently than CXCR4. Furthermore, hypoxia and serum starvation were required for cell surface display of CXCR4 and CXCL12 induction of ERK activation in HMEC-1 cells. In contrast, CXCR2 and CXCR3 mRNA levels remained, respectively, low and undetectable under all the conditions tested, and surface expression of CXCR2, CXCR3 and CXCR7 on the HMEC- 1 cells could not be demonstrated by FACS. In the human SK-MEL-5 melanoma cell line, CXCR4 mRNA expression was also increased under hypoxic conditions, whereas CXCR2 mRNA levels remained low and levels of CXCR3 and CXCR7 were undetectable. However, immunohistochemical staining of human metastatic melanoma sections demonstrated that CXCR2, CXCR3, CXCR4 and CXCR7 are expressed on tumor cells and, to a lesser extent, on endothelial cells. These results demonstrate that the tumor microenvironment regulates chemokine receptor expression through both cytokine and oxygen levels.     

Differential induction of cytokine genes and activation of mitogen-activated protein kinase family by soluble CD40 ligand and TNF in a human follicular dendritic cell line.

Follicular dendritic cells (FDC)3 play crucial roles in germinal center (GC) formation and differentiation of GC B cells. Many aspects of FDC function are influenced by contact with B or T cells, and by cytokines produced in the GC, which involve stimulation of CD40 and TNF-alpha receptors on FDC. In this study, using an established FDC line, HK cells, we compared the effects of CD40 and TNF receptor triggering on cytokine induction and activation of mitogen-activated protein kinase family. We show that HK cells spontaneously produced IL-6, M-CSF, and G-CSF mRNA. Both the soluble form of CD40 ligand (sCD40L) and TNF increased the level of M-CSF and G-CSF mRNA. While TNF strongly induced IL-6 mRNA, its expression was not affected by sCD40L treatment, differing from the strong IL-6 induction in other cell types upon CD40 stimulation. In addition, sCD40L treatment resulted in activation of extracellular signal-related kinase 1 and 2 (ERK1/2) and p38 without significant increase in c-Jun N-terminal kinase (JNK) activity. Lack of JNK activation differs in that most B cells respond to CD40 stimulation by inducing JNK activity strongly, suggesting distinct characteristics of CD40 signaling in FDC. Compared with the effects of sCD40L, TNF was capable of inducing JNK activity in addition to the activation of ERK1/2 and p38. Furthermore, the proximal signaling elements activated by TNF differed from those activated by sCD40L, in that TNF did not require PMA-sensitive protein kinase C isoforms in the activation of ERK and p38, whereas sCD40L did. However, signals activated by these stimuli converged on cytokine gene expression in a synergistic manner, which may have implication in augmenting FDC function during GC reaction.     

Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity.

Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ in molecular weight (58,000 and 73,000), and to be expressed differentially in different cells. It is further shown that polyclonal antibodies against one of the TNF binding proteins (TBPI) display, by virtue of their ability to bind the TNF receptor, activities which are very similar to those of TNF. These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies lack TNF-like activities, but acquire them upon cross-linking with anti-F(ab)2 antibodies, suggesting that the ability of the anti-TBPI antibodies to mimic TNF correlates with their ability to cross-link the TNF receptors. This notion was further supported by data obtained in a comparative study of the TNF-like cytotoxicity of a panel of monoclonal antibodies against TBPI. The induction of TNF-like effects by antibodies to a TNF receptor suggests that TNF is not directly involved in intracellular signalling. Rather, it is the receptors to this cytokine which, when properly triggered in a process which appears to involve clustering of these receptors, transduce the signal for response to TNF into the cell's interior.