Antagonistic control of tumor necrosis factor receptors by protein kinases A and C. Enhancement of TNF receptor synthesis by protein kinase A and transmodulation of receptors by protein kinase C.

We have investigated control mechanisms of TNF receptor expression (TNF-R) in various human tumor cells and normal peripheral blood monocytes. Activators of protein kinase A (PKA) signal transduction pathways were found to enhance TNF-R expression up to sevenfold, whereas in the same cells, IFN-alpha and -gamma receptors remained unaffected. Inhibitors of protein kinases downregulate both constitutive and cAMP-enhanced TNF-R expression. Binding studies revealed an increase in TNF-R numbers without a change in receptor affinity. Both, direct activators of PKA and inhibitors of phosphodiesterase, raising intracellular levels of cAMP, were found to be effective. As activation of PKA does not slow down the degradation rate of TNF-Rs, but rather enhances protein synthesis-dependent reexpression of TNF-Rs after transient PKC-mediated transmodulation and after tryptic digestion of TNF-Rs, it is concluded that PKA stimulates TNF-R synthesis. Maximum TNF-Rs enhancement is reached after 24 h of stimulation and is reversible, suggesting that receptor upregulation is not linked to irreversible steps of cellular differentiation. PKA-mediated enhancement of TNF-R expression was predominantly observed in normal peripheral blood monocytes and tumor cell lines of myeloid origin. As in these typical TNF producer cells, the production of TNF is also controlled by PKA and PKC, a regulatory circuit is proposed, by which these two independent signal pathways antagonistically regulate TNF production and, at the receptor level, TNF sensitivity.

Identification of a 60-kD tumor necrosis factor (TNF) receptor as the major signal transducing component in TNF responses.

We describe here a monoclonal antibody (H398) that immunoprecipitates a human 60-kD tumor necrosis factor (TNF) membrane receptor (p60) and competes with TNF binding to p60 but not to p85 TNF receptors. Despite partial inhibition of TNF binding capacity of cells coexpressing both TNF receptor molecules, H398 uniformly and completely inhibits very distinct TNF responses on a variety of cell lines. These data suggest a limited structural heterogeneity in those components actually contributing to TNF responsiveness and identify p60 as a common receptor molecule essential for TNF signal transduction. As H398 is a highly effective TNF antagonist in vitro, it might be useful as a therapeutic agent in the treatment of TNF-mediated acute toxicity.

Downregulation of tumor necrosis factor (TNF) sensitivity via modulation of TNF binding capacity by protein kinase C activators.

The regulatory action of activators for protein kinase C on the specific binding capacity for recombinant human tumor necrosis factor alpha (TNF-alpha) was studied on various human cell lines. Phorbol myristate acetate (PMA) and oleyl acetyl glycerol (OAG) both are able to rapidly downregulate TNF-binding capacity of normal and malignant cells derived from various tissues. As PMA treatment did not enhance internalization of TNF-alpha-receptor complexes at 37 degrees C, and since OAG was able to downregulate TNF-binding capacity under conditions where internalization and shedding of receptor protein are prevented, we conclude that protein kinase C controls ligand affinity of the TNF-receptor protein, possibly via direct phosphorylation. Protein kinase C triggered downregulation of TNF-alpha-binding capacity concomitantly resulted in reduction of TNF-alpha sensitivity, as revealed from decreased cytotoxic action of TNF-alpha on L 929 cells and from inhibition of TNF-alpha-mediated enhancement of HLA class II antigen expression in Colo 205 cells. Restoration of TNF-binding capacity upon abrogation of protein kinase C stimulation leads to full recovery of TNF responsiveness, further supporting the close linkage of TNF-receptor expression and TNF sensitivity. These data suggest that regulation of TNF-binding capacity by protein kinase C is one of the cellular control mechanisms of TNF responsiveness.

Alloreactive and H-2-restricted Lyt 23 cytotoxic T lymphocytes derive from a common pool of antecedent Lyt 123 precursors.

If the collaborative requirement of Lyt 1 T helper cells is bypassed by the Lyt 1 T cell-derived mediator of T help, termed Il-2, upon antigenic stimulation, PNA+ Lyt 123 thymocytes differentiate into either alloreactive or H-2-restricted PNA- Lyt 23 cytotoxic effector cells. Along the differentiation pathway from Lyt 123 leads to 23 effector cells, cytolytic activity is carried out by T cells that still express the Lyt 123 phenotype. The data establish that Lyt 23 CTL are produced by differentiation from antecedent Lyt 123 cells.

Virus and trinitrophenol hapten-specific T-cell-mediated cytotoxicity against H-2 incompatible target cells.

Immune spleen cells from LCM virus-infected (CBA X C57BL/6)F1 radiation chimeras entirely repopulated with CBA-T6 lymphocytes were cytotoxic for allogeneic, LCM virus infected C57BL/6 mouse-derived target cells. Normal C57BL/6 targets were not lysed. CBA-T6 lymphocytes derived from (CBA X C57BL/6) radiation chimeras sensitized in vitro against TNP-conjugated C57BL/6 spleen cells lysed TNP-conjugated C57BL/6 targets. However normal C57BL/6 mouse-derived targets were not destroyed. The magnitude of virus-specific (or TNP-specific) cytotoxic responses against H-2 incompatible targets was lower compared to that against H-2 compatible targets. These data are considered to support and to extend the altered self concept, but are not consistent with the dual recognition concept.

T-T cell interactions during in vitro cytotoxic T lymphocyte responses. V. Precursor frequencies and specificity of alloreactive helper T cells.

We assessed the quantitative representation and specificity of alloreactive helper T lymphocytes (HTL) within murine spleen cells by three different limiting dilution systems. For the induction of primary cytolytic T lymphocyte (CTL) responses towards alloantigens, a Lyt-1+23- HTL precursor (HTLp) could be defined, which occurred at frequencies of 1/2.000-1/50,000, depending on the alloantigen in question. The HTLp limiting for interleukin-2 (Il-2) production also expressed the Lyt-1+ phenotype and occurred in similar frequencies. This cell type was concluded to be the limiting HTLp for the overall helper activity required for the induction of primary CTL responses. HTLp reactive to Mlsa -encoded antigens occurred at higher frequencies (1/500) than those reactive towards whole allogeneic H-2 haplotypes (1/4,000-1/7,000). Within the H-2 complex, I region-encoded alloantigens activated approximately 10 times more HTLp than did H-2K or H-2D regions. When alloreactive HTL were tested for antigen specificity at the clonal level, approximately 80% of the HTL clones proved to be specific to the alloantigen used for immunization, whereas approximately 20% reacted also towards third-party alloantigens. The data are discussed with respect to putative T-T interactions within the helper T cell population and the precision of alloantigen recognition by HTL.

Lyt-23+ cyclophosphamide-sensitive T cells regulate the activity of an interleukin 2 inhibitor in vivo.

Sera of thymus-bearing normal mice contain high levels of Interleukin 2 (II-2) inhibitor, whereas sera of athymic nu/nu mice do not. Evidence is presented that cyclophosphamide-sensitive Lyt-23+ T cells induce high II-2 inhibitor activity in the recipient nu/nu mice in the course of a graft-vs.-host reaction. The II-2 inhibitor has an approximately 50,000 mol wt. Its function is neither antigen specific nor H-2 restricted. During ontogeny, its activity parallels the development of T cell reactivity, i.e., it is absent both in the amniotic fluid and in sera of unborn mice, but increases to high levels during the early postnatal phase. The II-2 inhibitor described is viewed as an example of a T cell-dependent, in vivo regulatory mechanism able to effectively counteract the nonspecific activity of the Lyt-1+ helper T cell-derived II-2. Because the II-2 inhibitor activity is rather high in vivo, II-2 activity will exist only in close proximity to its producer cell, thereby maintaining specificity during the in vivo induction of cytotoxic T lymphocytes

H-2 restriction as a consequence of intentional priming. Frequency analysis of alloantigen-restricted, trinitrophenyl-specific cytotoxic T lymphocyte precursors within thymocytes of normal mice.

An in vitro acute-depletion protocol was used to detect trinitrophenyl (TNP)-specific, allo-major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) within thymocytes of inbred mice. After removal of alloreactivity, the negatively selected cells could be sensitized to become TNP-specific, allo-MHC-restricted cytotoxic T cells. A precursors frequency analysis revealed a three- to ninefold lower frequency of allo-MHC-restricted CTL precursors (CTL-P) as compared to self-MHC-restricted CTL-P. The specificity analysis of clonally distributed allo-MHC-restricted CTL-P excluded cross-reactivity as an explanation of allo-MHC restriction. These results provide direct evidence that thymic T cells are composed of a mixture of self-MHC- and allo-MHC-restricted immunocompetent T cells and that antigen-driven selection of precommitted T cells dictates the H-2-restriction phenotype, i.e., H-2 restriction is a consequence of priming.

Cyclophosphamide-sensitive T lymphocytes suppress the in vivo generation of antigen-specific cytotoxic T lymphocytes.

Murine T lymphocytes sensitized in vitro against either allogeneic lymphocytes or syngeneic hapten-conjugated lymphocytes do differentiate into highly effective cytotoxic T lymphocytes (CTL) (1-3). In vivo immunization of T lymphocytes to the same antigens, however, results in the generation of only marginal cytotoxic activity (1,4,5). Recently we found that the weakness of in vivo generated cytotoxicity is not due to a failure of antigen-induced T-cell sensitization but rather due to suppression of the in vivo differentiation of sensitized CTL precursors into effective CTL(6). In keeping with this finding it was postulated that suppressor cells may regulate the in vivo differentiation of CTL. We now report, that cyclophosphamide-sensitive T cells suppress the in vivo differentiation of antigen-specific CTL. Thus, pretreatment of mice with a single dose of cyclophosphamide (100 mg/kg) converts their state of low responsiveness to a state of high responsiveness.

Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells.

Tumor necrosis factor (TNF) and lymphotoxin (LT) are highly pleiotropic cytokines that play a central role in regulating HIV-1 replication. These cytokines express their activities through two membrane receptors, TNFR60 (p55-60) and TNFR80 (p75-80). In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production. Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells. More relevant, in vitro HIV-infected peripheral blood lymphocytes cocultured with cells expressing membrane TNF underwent rapid induction of apoptosis with a subsequent reduced HIV production of these lymphocytes cultures. This was not observed with HIV-infected lymphocytes treated with soluble TNF. These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

Dissection of the proliferative and differentiative signals controlling murine cytotoxic T lymphocyte responses.

Evidence is presented that interleukin 2 (IL-2) is not sufficient to cause the differentiation of primary cytotoxic T lymphocytes (CTL). Sources of IL-2 were compared for their ability to cause proliferation as well as differentiation into CTL. Whereas all factors caused proliferation, only the crude Con A supernatant had cytotoxic T cell differentiation factor (CTDF) activity. Furthermore, factors absorbed with an IL-2-dependent cell line to remove IL-2 still retained CTDF activity. Thus, IL-2 functions to cause clonal expansion of CTL precursors preactivated by antigen or mitogen, but for their differentiation into CTL, an additional factor is required, here called CTDF.

Frequency analysis of cytotoxic T lymphocyte precursors in chimeric mice. Evidence for intrathymic maturation of clonally distinct self-major histocompatibility complex- and allo-major histocompatiblilty complex-restricted virus-specific T cells.

To study whether the thymic major histocompatibility complex (MHC) imposes a constraint on the receptor repertoire of maturating cytotoxic T lymphocyte (CTL) precursors, the restriction phenotypes of virus-specific CTL of MHC-compatible and of MHC-incompatible thymus- and bone marrow-grafted (A X B)F1 chimeric mice were compared. Dependent on the mode of in vitro sensitization, thymocytes or splenocytes of both types of chimeric mice generated Sendai virus-specific, self-MHC-or allo-MHC-restricted CTL. By applying the limiting-dilution technique, the CTL-precursor (CTL-P) frequencies of self-MHC-restricted and allo-MHC-restricted virus-specific T cells as well as of alloreactive T cells were determined. The data obtained revealed that independent of MHC differences between thymus and bone marrow, the frequencies of self-MHC-restricted and allo-MHC-restricted CTL-P were comparable, and in the same older of magnitude as those previously determined in conventionally reared mice. Self-MHC-restricted, virus-specific CTL-P were in a three- to fivefold excess over allo-MHC-restricted CTL-P. A segregation analysis revealed that clonally distinct CTL-P give rise to either self-restricted or allo-MHC-restricted, virus-specific CTL. Both sets were found not only in the spleen, but also in the thymus of chimeric mice, formally demonstrating the intrathymic differentiation pathway of self-MHC as well of allo-MHC-restricted CTL-P. These data reveal no major constraint of the thymic MHC on the capacity of T cells to recognize viral antigens either in the context of self-MHC or of allogeneic MHC products.

Protein kinase C mu is located at the Golgi compartment.

Protein kinase C mu (PKC mu) displays unusual structural features like a pleckstrin homology domain and an amino-terminal hydrophobic region with a putative leader peptide and transmembrane sequence. As a discrete location often is a direct clue to the potential biological function of a kinase, antibodies directed against unique amino- and carboxy-terminal domains of PKC mu were used to localize the protein within intracellular compartments in immunofluorescence and subcellular fractionation studies. Confocal laser scanning microscopy showed colocalization of PKC mu with the resident Golgi marker protein beta 1,4 galactosyltransferase in PKC mu transfectants and in the human hepatocellular carcinoma cell line HepG2, expressing endogenous PKC mu. Long-term treatment of cells with brefeldin A, which disintegrates the Golgi apparatus, disrupted PKC mu-specific staining. Cosegregation of PKC mu with beta 1,4 galactosyltransferase, but not with the endosomal marker rab5, upon density gradient fractionation and Western blot analysis of HepG2 cell extracts, provides independent evidence for a Golgi localization of PKC mu. Moreover, cellular sulfate uptake and Golgi-specific glycosaminoglycan sulfation was enhanced in PKC mu transfectants. Together, these data suggest that PKC mu is a resident protein kinase of the core Golgi compartment and is involved in basal transport processes.

Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L.

Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity.

Activation of CD95L fusion protein prodrugs by tumor-associated proteases.

To achieve tumor cell-restricted activation of CD95, we developed a CD95L fusion protein format, in which CD95L activity is only unmasked upon antibody-mediated binding to tumor cells and subsequent processing by tumor-associated proteases, such as matrix metalloproteases (MMPs) and urokinase plasminogen activator (uPA). On target-negative, but MMP- and uPA-expressing HT1080 tumor cells, the CD95L prodrugs were virtually inactive. On target antigen-expressing HT1080 cells, however, the CD95L prodrugs showed an apoptotic activity comparable to soluble CD95L artificially activated by crosslinking. CD95 activation by the CD95L prodrugs was preceded by prodrug processing. Apoptosis was blocked by inhibitors of MMPs or uPA and by neutralizing antibodies recognizing the targeted cell surface antigen or the CD95L moiety of the prodrugs. In a xenotransplantation tumor model, local application of the prodrug reduced the growth of target antigen-expressing, but not antigen-negative tumor cells, verifying targeted CD95L prodrug activation in vivo.

Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug.

Tumor necrosis factor (TNF) prodrugs are fusion proteins comprised of an N-terminal single-chain antibody variable fragment (scFv) targeting a TNF effector and a C-terminal TNF receptor (TNFR)1-derived inhibitor module. Introduction of matrix metalloproteinase (MMP)-2 recognition motifs between TNF and the TNFR1 fragment allowed activation by recombinant MMP-2 and MMP-expressing HT1080 cells. Processing by endogeneous MMPs required specific membrane binding of the TNF prodrug via the targeting scFv, ensuring strictly antigen-dependent activation. Interestingly, TNF bioactivity of the processed prodrug was approximately 1000-fold higher upon scFv-mediated targeting, and signaled juxtatropic cell death also to antigen-negative cells. Microscopical analyses of TNFR2 clustering and TNF receptor-associated factor 2 recruitment at contact sites to adjacent cells revealed the formation of stable TNFR complexes by target-bound, processed prodrug, resembling the increased signal capacity of natural, membrane-expressed TNF. MMP-2-sensitive TNF prodrugs represent novel cytokine-based reagents for targeted cancer therapy, which should be exploitable for MMP-overexpressing tumors.

Modulation of monocyte activation in patients with rheumatoid arthritis by leukapheresis therapy.

One of the hallmarks in rheumatoid arthritis (RA) is the intense activation of the monocyte-macrophage system. In the present investigation, the modulation of blood monocyte activation was studied with regard to the secretion of cytokines and inflammatory mediators, and to the expression of cytokine receptors. Patients with severe active RA underwent repeated leukapheresis procedures that removed all circulating monocytes. Highly enriched monocyte preparations from the first and third leukapheresis were studied. There were striking differences between the two monocyte populations. Cells obtained from the first leukapheresis constitutively released large amounts of prostaglandin E2 (PGE2), neopterin, interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). In particular, IL-1 beta and neopterin production were further enhanced by stimulation with either interferon-gamma (IFN-gamma) or TNF-alpha without a synergistic effect. In contrast, cells derived from the third leukapheresis procedure showed a close to normal activation status with only low levels of cytokine and mediator production as well as a reduced response to cytokine stimulation. The number of the receptors for IFN-gamma and TNF-alpha was not changed between first and third leukapheresis. However, TNF-binding capacity was only detectable upon acid treatment of freshly isolated monocytes. A further upregulation was noted upon 24 h in vitro culture, suggesting occupation of membrane receptors and receptor down-regulation by endogenously produced TNF-alpha. Northern blot analysis of cytokine gene expression was in good correlation with the amount of mediators determined on the protein level. These data indicate that cells of the monocyte-macrophage system are already highly activated in the peripheral blood in RA patients with active disease. These cells can be efficiently removed by repeated leukapheresis and are replenished by monocytes that have, with respect to cytokine and mediator production, a considerably lower activation status.

Tumor necrosis factors in 1996.

The 6th International Congress on Tumor Necrosis Factors and Related Molecules was held in Faliraki, Island of Rhodes, Greece, 8-12 May, 1996. This review summarizes the topics addressed and highlights some of the major advances presented during the meeting.

Sensitivity of leukemia cell lines to cytotoxic alkyl-lysophospholipids in relation to O-alkyl cleavage enzyme activities.

The human leukemia cell lines K562, HL60, and Raji and the mouse leukemia cell line L1210 showed a differential susceptibility to the action of the alkyl-lysophospholipid (ALP) 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). After 48 hours, the 50% growth-inhibition doses (ID50) of ET-18-OCH3 were found to be 0.78 microgram/ml (HL60), 1.53 microgram/ml (Raji), 4.41 micrograms/ml (K562), and 5.05 micrograms/ml (L1210), as determined by [3H]thymidine incorporation. At the same time, cell viability was determined by trypan blue exclusion and revealed median lethal doses (LD50) of 3.5 micrograms/ml (HL60), 15 micrograms/ml (Raji), 24 micrograms/ml (L1210), and 38 micrograms/ml (K562). Since O-alkyl cleavage enzyme previously was suggested as being important in the detoxification of cytotoxic ALPs, the enzyme activity was compared with the susceptibility to ET-18-OCH3 in the distinct cell lines. In comparison to an approximate sevenfold to elevenfold (ID50 and LD50, respectively) difference in the susceptibility of the above leukemia cell lines to ET-18-OCH3, no significant difference in the specific activities (0.13-0.21 nmol/min/mg) of the O-alkyl cleavage enzyme was found in the above leukemia cell lines. Therefore, the differential sensitivity of the above lines investigated cannot be explained by differences in O-alkyl cleavage enzyme activity. Experiments with radiolabeled ET-18-OCH3 in Raji cells suggest, rather, a critical role for phospholipases C and/or D in ALP metabolism.

Tumor necrosis factor and lymphotoxin gene expression in human tumor cell lines.

In this paper we show that eight of 17 tumor cell lines of various tissue origin constitutively express tumor necrosis factor (TNF) mRNA. Five of these eight cell lines concomitantly contained lymphotoxin (LT) mRNA. Of the remaining nine cell lines that lacked detectable TNF or LT gene expression, five could be induced by phorbol ester and/or cytokines to accumulate the respective mRNAs. While TNF mRNA was found not only in neoplastic hematopoietic cells, but also in cell lines derived from carcinomas, LT gene expression seemed to be restricted to lymphoid tumor cells. Tumor cells that expressed LT mRNA also secreted LT protein and proved to be resistant to the cytostatic/cytotoxic effects of their own protein product as well as to exogenous recombinant TNF and recombinant LT. In contrast, most of the cell lines containing TNF mRNA did not release TNF protein into the supernatants, indicating that TNF gene expression may be controlled predominantly at a post-transcriptional level. Thus, the presence of TNF mRNA does not necessarily reflect a TNF-resistant phenotype. Our findings demonstrate that TNF and/or LT mRNA expression is a rather common phenomenon in long-term cultured tumor cell lines and reveal that ectopic TNF and/or LT production by tumor cells may be involved in certain paraneoplastic syndromes as well as in tumorigenesis.