The role of receptor-ligand endocytosis and degradation in interleukin-2 signaling and T-lymphocyte proliferation.

The specific intracellular signaling pathways for interleukin-2 (IL-2) that lead to delivery of the proliferative stimulus are currently unknown. We and others have excluded signaling pathways used by other growth factors and by the antigen-specific T-cell receptor, such as increased intracellular Ca2+ concentrations, activation of protein kinase C, or ion transport across the plasma membrane. One feature of IL-2 signaling that may be important in delivery of the proliferative stimulus is endocytosis and processing of the lymphokine receptor-ligand complex. In this study we examined these steps in receptor signaling by mouse CTLL-2 cells and human OKT3-activated T-cells using monoclonal antibodies specific for the 55 kDa alpha-subunit of the IL-2R that allow IL-2 binding but block endocytosis, and with lysosomotrophic amines that selectively inhibit receptor mediated endocytosis and/or processing of IL-2. Our results demonstrate that these inhibitors block receptor endocytosis, ligand degradation, c-fos protooncogene activation, and ultimately proliferation of the IL-2-dependent T-cell line, CTLL-2. In heterogeneous populations of activated human T cells the lysosomotrophic amines demonstrated a greater inhibition of degradation than of endocytosis. These observations support the hypothesis that IL-2/IL-2R endocytosis and ligand/receptor processing or degradation may be important steps in lymphokine signal transduction.

Lactational alcohol exposure elicits long-term immune deficits and increased noradrenergic synaptic transmission in lymphoid organs.

Increasing evidence suggests that the sympathetic nervous system plays an important role in immunomodulation. While chronic alcohol consumption has been associated with immune deficits, the effects of exposure to alcohol during early postnatal life on subsequent immunocompetence and activity of sympathetic neurons in lymphoid organs are not known. This study examined the long-term effects of lactational alcohol consumption on cellular immune responses and noradrenergic synaptic transmission in lymphoid and other organs of the young adult C57BL/6 mouse. The data show that exposure to alcohol via the mother's milk was associated with long-term deficits in cellular immunity, including suppression of the local graft vs host and contact hypersensitivity responses. The animals also displayed enhanced noradrenergic synaptic transmission and decreased beta-adrenoceptor density selectively in lymphoid organs. These neuroimmune changes are particularly striking since body weight-gain of the suckling pups was normal and their blood alcohol concentration was considerably lower than that of the alcohol-consuming dam. This suggests an increased sensitivity of the nascent immune and nervous systems during the critical period of early postnatal development.

Prenatal ethanol exposure alters immune capacity and noradrenergic synaptic transmission in lymphoid organs of the adult mouse.

Clinical and experimental evidence indicates that exposure to alcohol in utero is associated with altered immune capacity. The mechanisms underlying such abnormalities are not clear. However, the suggestion that reciprocal interactions between the immune and the nervous systems are necessary for a competent immune response may be relevant. This work examined the consequences of prenatal ethanol exposure on cellular immune responses and noradrenergic synaptic transmission in lymphoid organs of the adult C57B1/6 mouse. Pregnant mice were fed a liquid diet containing 25% of the calories as ethanol (4.8% w/v) or pair-fed an isocaloric equivalent of this diet throughout gestation, followed by foster-nursing the neonates on normal dams. As young adults, mice exposed to ethanol prenatally displayed immunologic and selective neurochemical changes: (1) depressed ability to produce cellular immune responses, including contact hypersensitivity and a local graft-vs-host response, and (2) altered noradrenergic synaptic transmission, including enhanced norepinephrine turnover, and a reduction in norepinephrine levels and beta-adrenoceptor density in the thymus and spleen, but not the heart. However, both the integrity and compartmentation of noradrenergic nerve fibres in the spleen were intact. It is suggested that altered noradrenergic synaptic transmission selectively in lymphoid organs may contribute to the impaired immune capacity associated with fetal alcohol exposure.

Afferent and efferent specificity in the induction and elicitation of parental cross-protective immunity by an immunogenic murine tumor variant: associative recognition of a unique tumor-specific antigen on somatic cell hybrids.

Highly immunogenic (Imm+) murine tumor cell variants can engender a strong tumor-specific, cross-protective immune response against challenge with the weakly immunogenic parental tumor cell line. We examined the afferent induction and efferent specificity of the parental cross-protective immunity observed following immunization with the Imm+ variant of the murine fibrosarcoma MCA-F, designated MCA-FM1. Specificity of the afferent and efferent responses against the parental tumor in mice immunized with the MCA-FM1 variant were monitored by challenge with the tumor MCA-D, which expresses a tumor-specific antigen that is immunologically distinct from but biochemically related to the MCA-F antigen. We observed that mixture of MCA-D and MCA-FM1 cells at immunization failed to elicit a strong tumor rejection response against challenge with MCA-D. Challenge of MCA-FM1-immune mice with a mixture of MCA-FM1 and MCA-D cells resulted in a significant bystander effect at the site of Imm+ rejection, with reduced growth of the MCA-D tumor. To test the hypothesis that the induction of parental cross-protective immunity required the associative recognition of both the Imm+ neoantigen and the parental tumor antigen on the same cell, we constructed somatic cell hybrids of MCA-D with either MCA-F or MCA-FM1. Surprisingly, the hybrids did not express either parental tumor-specific antigen present on the fusion partners but displayed a unique antigenic specificity designated F/D. Expression of the F/D antigen by both the immunogenic and nonimmunogenic hybrid cell lines demonstrated that the tumor-specific F/D antigen was the focus of the cross-protective immunity. These results demonstrate that associative recognition of the tumor-specific parental antigen with the strongly immunogenic neoantigen coexpressed on the surface of the Imm+ variant is responsible for the afferent induction and efferent elicitation of anti-parental cross-protective immunity. Furthermore, this study is the first to report that the fusion of two syngeneic tumor cell lines reproducibly results in a new tumor antigen specificity at the expense of the original parental specificities.

Immunological characteristics of immunogenic variants induced in the MCA-F murine fibrosarcoma using 1-methyl-3-nitro-1-nitrosoguanidine, 5-aza-2'-deoxycytidine, or ultraviolet radiation.

The purpose of this study was to compare the frequency of generation and in vivo cross-reactivity of highly immunogenic (Imm+) clones induced in a single parental murine fibrosarcoma cell line MCA-F by 4 weekly treatments with either UV-B radiation, 1-methyl-3-nitro-1-nitrosoguanidine, or 5-aza-2'-deoxycytidine. These agents are believed to induce Imm+ variants by different mechanisms. The frequency of Imm+ variant generation was similar for the three different protocols, suggesting that the frequency of Imm+ generation was related more closely to the cell line than the inducing agent used. The strength of the immunogenic phenotype, however, was better correlated to the agent used, since 1-methyl-3-nitro-1-nitrosoguanidine yielded clones with the strongest immunogenicities. Three of four UV-B-induced Imm+ clones grew preferentially in chronically UV-irradiated syngeneic mice, a phenotype associated with UV-induced skin tumors. Cross-reactivity was tested with two Imm+ clones from each treatment group in a modified immunoprotection assay that selectively engendered antivariant, but not antiparental, immunity. Under these conditions each clone, except one, protected against itself. The clones displayed a complex pattern of cross-protection. Intervariant cross-protection was sensitive to the challenge dose, suggesting possible differences in the strengths of the cross-reacting immunities. Conversely, parental cross-protection was observed only with high immunizing multiplicities of Imm+ cells. The clones expressed the Imm+ phenotype in both C3H/HeN and C3H/HeJ mice, suggesting that expression of mammary tumor virus antigens did not account for the strong antitumor immune response. We also investigated whether the level of major histocompatibility complex class 1 or class 2 expression and immunogenic phenotype were correlated. Flow cytofluorography using haplotype-specific anti-Kk and anti-Dk monoclonal antibodies did not reveal a consistent difference in the constitutive or gamma-interferon-induced class 1 expression by Imm+ clones. However, we did observe a significant increase in the constitutive expression of IAk by most of the Imm+ variant clones. Together, these data demonstrate that in this system Imm+ variants engendered by a variety of mechanisms can express a range of cross-reactive tumor rejection neoantigens, independent of parental tumor antigens or major histocompatibility complex antigen expression.

Low-molecular-weight membrane component inhibits the metastatic phenotype of B16-F10 melanoma.

Treatment of the metastatic melanoma cell lines B16-F1 and B16-F10 with 1.5-2 per cent butanol elevates their experimental metastatic potential, whereas reconstitution of butanol-extracted B16 cells with crude butanol extracts decreases the number of experimentally induced lung foci. We partially purified the biologically active components from crude butanol extracts of B16-F1 by high-performance liquid chromatography and isoelectric focusing, and found the inhibitory activity (i) was in the low-molecular-weight (5-10 kDa) fraction of the chromatogram, (ii) had an isoelectric pH between 5.6 and 5.8, (iii) was distinct from a thiol-protease activity eluted from the isoelectric focusing bed at pH 4.9-5.3 and (iv) was not itself an inhibitor of serine or thiol proteases. Incubation of butanol-extracted B16-F10 cells with known inhibitors of serine, acid and thiol protease inhibitors had no effect on the experimental metastatic phenotype. Although the apparent molecular weight was low, the inhibitor(s) tended to aggregate after focusing, probably owing to the presence of carrier ampholines. Using two-dimensional gel electrophoresis, we observed slight differences between intact and butanol-extracted cells, most of them in the low-molecular-weight region. These results suggest that butanol treatment may reversibly release certain inhibitors of cell surface enzymes other than proteases, which might be involved in invasion and metastasis.

Isolation and in vitro characterization of a novel immunosuppressive cyclosporine metabolite.

A novel metabolite (M-E) was identified by high-performance liquid chromatography in the serum of cyclosporine-treated renal transplant recipients during a second wave of immunosuppressive activity after disappearance of the initial wave due to the direct effect of CsA. M-E was identified in human serum and porcine bile both by HPLC and by a preparative thin-layer chromatography (TLC). It demonstrated homogeneity with characteristic retention times on C8 and C18 column HPLC systems using a variety of elution systems, and distinctive TLC mobility (Rf 0.35). Metabolite E (M-E) was documented to be a CsA metabolite by radioactive tracer studies, by crossreactivity with a polyclonal sheep antibody in radioimmunoassay, and by the presence of a characteristic mass spectrum. Further, in vitro immunosuppressive assays documented effects of M-E similar to those of CsA. The relative activity of M-E versus CsA was quantitated by potency ratios: for inhibition of normal human mixed lymphocyte culture reactions, the ratio was 0.79 +/- 0.23. Interindividual differences were observed in patient susceptibility to MLR inhibition not only by CsA, as previously reported by others, but also by M-E. There was a lesser effect of M-E compared with CsA in inhibiting proliferation of, and IL-2 generation by, C3H murine splenocytes stimulated with concanavalin A: the potency ratios for both systems were about 0.5, possibly reflecting an interspecies variability in generation of or susceptibility to M-E. These studies suggest that heretofore unidentified metabolites--including, but not limited to, M-E--may play an important role in the immunosuppressive effect of CsA in man.

Characterization of variant and parental-cross-protective immunity to immunogenic variants of a murine fibrosarcoma using the local adoptive transfer assay.

The purpose of this study was to characterize the lymphocyte populations responsible for rejection of immunogenic (Imm+) tumor variants, and the cross-protective immunity engendered by Imm+ variants against the weakly immunogenic parental tumor. Immunogenic clones of the weakly immunogenic methylcholanthrene-induced fibrosarcoma MCA-F have been generated using 1-methyl-3-nitro-1-nitrosoguanidine, 5-aza-2'-deoxycytidine, or ultraviolet radiation (UV-B; 280-320 nm). These clones grow progressively in immunosuppressed adult-thymectomized irradiated mice, but are rejected by immunocompetent syngeneic hosts. The parental MCA-F tumor grows progressively in both groups. Mice that have rejected a challenge of 1 x 10(5) Imm+ cells show an anamnestic immune response against both the Imm+ clone and the parental MCA-F tumor. Using the local adoptive transfer assay and depletion of T-cell subsets with antibody plus complement, we show that immunity induced by the Imm+ variants against the parent MCA-F was mediated by the Thy1.2+, L3T4a+ population without an apparent contribution by Lyt2.1+ cells. Although antivariant immunity was also dependent upon Thy1.2+ cells, depletion of either the L3T4a+ or the Lyt2.1+ cells failed to abolish immunity against the variant. A role for Lyt2.1+ T lymphocytes in antivariant immunity, but not antiparent immunity, was supported by the results of cytotoxic T lymphocyte (CTL) assays. Following immunization with high numbers (1 x 10(5) to 5 x 10(5) of viable Imm+ cells, antivariant, but not antiparent CTL activity was detected in mixed lymphocyte tumor cell cultures. Immunization with lower numbers (3 x 10(4] of viable Imm+ or with high numbers of mitomycin-C-treated Imm+ engenders only antivariant immunity without parental cross-protection. Under these conditions lymphocytes mediating immunity against the variant in the local adoptive transfer assay were exclusively of the Thy1.2+, L3T4a+ phenotype, with no contribution from the Lyt2.1+ cells. Identical results were obtained for Imm+ clones of MCA-F induced by methylnitronitrosoguanidine, 5-azadeoxycytidine, and UV-B, suggesting that the nature of the antitumor immunity engendered by Imm+ is not significantly affected by the agent used. Furthermore, these results demonstrate that the cross-reactivity and cellular effectors of antitumor immunity in this system are influenced by the immunizing dose of Imm+ cells: the predominant effectors of both antivariant and parental-cross-reactive immunity were of the CD4+ T cell subclass, with a CD8+ cytotoxic population contributing to antivariant immunity only after high-dose immunization.

Inhibition of experimental metastasis and extracellular matrix degradation by butanol extracts from B16-F1 murine melanoma.

We previously demonstrated that noncytolytic butanol extraction of B16 melanoma cells can increase the number of experimental lung metastases, and that brief incubation of the extracted cells with the extracted moieties reduces metastatic phenotype. This study examined the possibility that the extracted components are endogenous inhibitors of tumor cell surface-associated, degradative enzymes. The activity was found to be tumor associated, since only tumor extracts could reduce the number of experimental lung metastases of a variety of solid tumors. The activity in crude butanol extracts of B16-F1 that modulated the metastatic phenotype of extracted B16-F10 was partially purified by preparative isoelectric focusing and high-performance gel permeation chromatography. Incubation of extracted B16-F10 cells with low (Mr 2,000-10,000) molecular weight materials focusing in the pH 5.6 to 5.8 region of the preparative isoelectric focusing gradient significantly reduced the number of experimental lung foci. Ampholines alone had no effect. Evidence that the extracted moiety might be an endogenous enzyme inhibitor was obtained with the use of the subendothelial matrix degradation assay, wherein B16-F10 cells digest 35S-labeled heparan sulfate proteoglycan. The same materials that reduced the metastatic potential of butanol-extracted B16-F10 cells also inhibited extracellular matrix degradation by 30 to 85%, as well as the activity of partially purified heparanase (endo-beta-glucuronidase). The metalloproteinase inhibitor 1,10-phenanthroline and the heparanase inhibitor heparin partially (30 to 50%) blocked extracellular matrix degradation. Conversely, inhibitors of serine, thiol, acid, and other proteases had little or no effect on extracellular matrix degradation. These data provide evidence that an endogenous, heat-stable inhibitor of cell surface degradative enzymes such as heparanase may play a role in hematogenous metastasis, and support the hypothesis that butanol extraction activates some of these surface enzymes by removing the endogenous inhibitors.

Differential extraction of tumor-specific antigens from two ultraviolet light-induced murine fibrosarcomas with the use of 1-butanol.

The purpose of this study was to investigate the immunobiological characteristics of the tumor-specific cell surface antigen expressed by the UV-induced murine fibrosarcoma, UV-2240. UV-2240 is classified as a regressor UV tumor because it is immunologically rejected by normal syngeneic mice but grows in immunocompromised or UV-irradiated hosts. The strong tumor-specific rejection antigen expressed by UV-2240 was found on the plasma membrane, and unlike the previously characterized antigen of UV-1591, the UV-2240 antigen was removed by using the noncytolytic butanol extraction technique. The tumor antigen activity in butanol extracts was resistant to digestion by endoglycosidase F and alpha-mannosidase, but was destroyed by pronase. In addition, the immunoprotective activity in extracts of UV-2240 was thermostable. These data demonstrate that the UV-2240-specific tumor antigen possesses physicochemical properties distinct from those of its well-characterized counterpart UV-1591.

Identification of cell surface cathepsin B-like activity on murine melanomas and fibrosarcomas: modulation by butanol extraction.

Cathepsin B (CB) is a lysosomal cysteine protease that may play a role in the activation of extracellular degradative enzymes involved in the destruction of the subendothelial matrix and extravasation of metastatic tumor cells. In this study we have investigated the cell surface expression of a CB-like enzyme on the surface of tumor cell variants expressing both high and low metastatic potentials. Cell surface CB-like activity was demonstrated by incubation of intact viable cells and isolated plasma membranes with the selective chromogenic substrate N-carbobenzoxyvalyllysyllysylarginyl-4-methoxy-beta-naphthylamide. Cell surface CB activity required thiol activation and was blocked by the CB-selective protease inhibitors leupeptin, antipain, and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by inhibitors inactive against CB. Enzymatic activity was significantly reduced when assayed at pH 7 and greater. Although all tumor lines had detectable CB-like activity, we observed a correlation between the expression of cell surface CB-like activity and metastatic phenotype only with isolated plasma membranes, and not with whole cell preparations. Noncytolytic 2% butanol extraction, a technique known to increase the experimental metastatic propensity, also significantly increased cell surface CB-like activity. Incubation of extracted tumor cells with crude butanol extracts prepared from those cells restored the cell surface CB-like activity to that of the unextracted controls, suggesting that the increased enzyme activity observed following extraction may be due to the release of an endogenous cysteine protease inhibitor. These results demonstrate that a CB-like protease is expressed on the surface of several murine tumor cells and that an endogenous inhibitor may play a role in determining experimental metastatic phenotype.

Inhibition of T-lymphocyte proliferation by the cyclic polypeptide didemnin B: no inhibition of lymphokine stimulation.

We investigated possible mechanisms by which the cyclic depsipeptide didemnin B (DB) inhibits lymphocyte proliferation. DB inhibited the proliferation of Con-A stimulated murine splenocytes, the interleukin-2 (IL-2) dependent proliferation of the CTLL-2 cell line, and the interleukin 4 (IL 4) dependent growth of both CTLL-2 and D10.G.4.1 cell lines at approximately equimolar concentrations (SD50 = 3 to 10 X 10(-9)M). Inhibition of CTLL-2 growth by 10(-8)M DB was partially reversible, and significantly blocked the incorporation of [3H]-thymidine even when added 24 hr after initial IL 2 stimulation. Concentrations of DB (10(-8)M) that completely blocked mitogen-driven spleen cell blastogenesis only partially inhibited the synthesis and secretion of IL-2. Although DB blocked the growth of T lymphocyte clones in response to both recombinant human IL 2 and recombinant murine IL 4, the suppression was not due to an uncoupling of lymphokinetic signaling but was closely correlated with an inhibition of protein and RNA synthesis. Addition of DB to an in vitro translation system did not inhibit protein synthesis. Thus, we conclude that DB functions as an antiproliferative, and not as a specifically immunosuppressive, compound.

Does interleukin 2 stimulus-response coupling result in generation of intracellular second messengers?

The binding of interleukin 2 (IL 2) to specific cell surface receptors provides a unique proliferative stimulus to sensitive T-lymphocytes. The purpose of this investigation was to examine the hypothesis that IL 2 stimulus-response coupling in the IL 2-dependent murine T-lymphocyte clone CTLL-2 employed some of the intracellular second messengers used by other growth factors. No evidence was obtained to implicate changes in intracellular Ca2+ concentrations, protein kinase C activation, or stimulation of the Na+/H+ antiporter or the Na+/K+ ATPase as requirements for stimulation by recombinant human IL 2. Pertussis toxin did not inhibit IL 2-driven growth of CTLL-2, and while cholera toxin did inhibit growth, its effect was optimal 6 to 8 hr after addition of IL 2 and could be mimicked by increased intracellular cyclic-AMP. Thus, guanine nucleotide-binding regulatory proteins do not appear to be involved in stimulation by this lymphokine. Together, these data suggest that IL 2 may not use any of the same types of intracellular second messengers generated subsequent to the binding of antigen or mitogen by T-lymphocytes.

Immunogenic variants of a murine fibrosarcoma induced by mutagenesis: requirement of viable cells for antigen-specific cross-protection.

The purpose of the study was to investigate the immunological and biological consequences of neoantigen expression by immunogenic tumor variants (Imm+) following in vitro treatment with the mutagen 1-methyl-3-nitro-1-nitrosoguanidine. The weakly immunogenic murine fibrosarcoma MCA-F was used because we have previously characterized the tumor-specific transplantation antigen expressed by this tumor. Immunogenic variant clones were obtained at high frequency following four treatments with 1-methyl-3-nitro-1-nitrosoguanidine. The immunogenicity of the Imm+ clones was confirmed by their progressive growth in immunosuppressed C3H/HeN mice and their lack of growth in normal syngeneic (C3H/HeN mice. The immune response engendered in immunocompetent mice after a single immunization with viable Imm+ cells was tumor specific, completely protecting hosts against challenge with 10,000-fold the minimum tumorigenic dose of parental MCA-F cells, but not against 10 minimum tumorigenic doses of the non-cross-reactive tumor MCA-D. The strong cross-protection elicited by Imm+ neoantigens against the parental tumor-specific transplantation antigen was not observed when soluble extracts or isolated plasma membranes of Imm+ cells were used for immunization. Immunogenic variant cells inactivated using either mitomycin C or gamma-irradiation also demonstrated a significantly diminished immunoprotective activity against challenge with the parent tumor. However, inactivated Imm+ cells and their isolated plasma membranes still expressed sufficient neoantigen to completely protect mice against homotypic Imm+, but not parental challenge. These results suggest that (a) the MCA-F Imm+ variants express neoantigens capable of engendering a strong specific as well as cross-protective immunity against challenge with either the parent or the variant and (b) the associative recognition of neoantigen and TSTA that results in strong cross-protection against challenge with the parent tumor requires immunization with viable Imm+ cells for full expression of the immunogenic phenotype.

Interleukin-2 stimulus-response coupling is calcium independent.

This study investigated the role of calcium (Ca2+) as a second messenger in the stimulus-response coupling of interleukin 2 (IL 2) binding to its specific receptor that results in lymphocyte proliferation. Although the Ca2+ channel blockers verapamil, nifedipine, and diltiazem induced a dose-dependent inhibition of [3H]-thymidine incorporation by HT-2 cells in response to recombinant human and purified rat IL 2, the stimulation indices of the treated and untreated cells were equivalent. Stimulation of HT-2 cells with recombinant human IL 2 (rIL 2) did not result in an increase in the concentration of intracellular free Ca2+ [( Ca2+]i), using the fluorescent intracellular Ca2+ indicator Fura-2AM. Conversely, partially purified rat IL 2 did cause an increase in [Ca2+]i that was not inhibited by the channel blockers or by chelation of extracellular free Ca2+. Thus, contaminating components in the partially purified rat IL 2, and not the IL 2 itself, resulted in increased [Ca2+]i by mobilization from intracellular stores. These results demonstrate that inhibition of lymphocyte proliferation by verapamil, nifedipine, and diltiazem is not due to an uncoupling of the IL 2 lymphokinetic signal, and stimulation of HT-2 cells using rIL 2 does not increase [Ca2+]i, and thus does not employ Ca2+ as a second messenger.

Comparison of the immunosuppressive effects of cyclosporine, lipid-soluble anesthetics, and calmodulin antagonists. Response to exogenous interleukin 2.

The purpose of these investigations was to compare the immunosuppressive mechanism of cyclosporine (CsA) with those of lipid-soluble local anesthetics and calmodulin antagonists. Chlorpromazine (CPZ) and pentobarbital (PB) both inhibit lymphocyte activation by attenuating sodium and potassium ion potentials. CPZ and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) can also block calcium-dependent activation processes by inhibition of calmodulin and protein kinase C. All four compounds were found to suppress human and murine lymphoproliferation to both alloantigen or mitogen in a dose-dependent and saturable manner. Exogenous interleukin-2 (IL-2) restored mitogenic responsiveness to cultures suppressed using W-7 and CsA, but not to lymphocytes suppressed with either CPZ or PB. Cytofluorographic analysis revealed that the degree of suppression in drug-treated lymphocytes was significantly correlated with the surface expression of receptors for transferrin and interleukin-2. Inhibition of IL-2 activation by PB was demonstrated to result from a blockade of the mitogenic growth factor signal using the IL-2-dependent cell line HT-2. Thus, the mechanism of action of cyclosporine can be differentiated from those of anesthetic immunosuppressants at the level of responsiveness to interleukin-2. The data support the hypothesis that cyclosporine may be an antagonist of calmodulin that selectively blocks early events in T lymphocyte activation leading to IL-2 synthesis, but does not inhibit the expression or function of the IL-2 receptor.

Does the binding of cyclosporine to calmodulin result in immunosuppression?

The cyclosporines are a family of cyclic endecapeptides that cause a profound suppression of primary immune stimulation both in vitro and in vivo. Recently, the regulatory protein calmodulin (CaM) has been implicated as a target for cyclosporin A (CsA) binding. This study utilized two less-active isomers of CsA to evaluate the specificity and biological significance of CaM binding. The three cyclosporines exhibited equivalent in vitro binding to CaM, regardless of immunosuppressive activity. Furthermore, CaM-dependent enzyme systems were inhibited equally by active and inactive cyclosporines, but only at concentrations 100 times those necessary to block lymphocyte activation. Thus the exquisite immunosuppressive stereospecificity displayed by cyclosporine isomers is not reflected in the binding to and inhibition of CaM, suggesting that inhibition of CaM-dependent processes is not sufficient to explain the immunosuppressive activity of CsA.

Biochemical characterization of 1-butanol-extracted murine tumor-specific transplantation antigens.

This investigation sought to characterize biochemically the tumor-specific transplantation antigens (TSTA) expressed on the cell surface of a panel of chemically induced fibrosarcomas of C3H/HeJ mice. Results suggest a uniform antigenic framework upon which individual specificities are superimposed. The antigens expressed by the 3-methylcholanthrene-induced fibrosarcomas MCA-D, MCA-F, and MCA-2A fulfill the requirements of a TSTA; namely, immunization of syngeneic hosts with irradiated cells or soluble extracts engenders a tumor-specific immune response such that animals resist challenge with the same, but not another, tumor. Brief incubation of intact tumor cells in single-phase aqueous solutions of 2.5% (v/v) 1-butanol extracts an immunoprotective TSTA, but not alloantigenic activity, from MCA-F cells. This extraction protocol was extended to the two other MCA-induced neoplasms. The butanol-extracted TSTA from the three tumors displayed isoelectric pHs of 6.4 to 6.6 following preparative isoelectric focusing. The tumor-specific immunoprotective activity from all three tumors displayed an apparent molecular weight of 150,000 (150 kDa) during high-performance gel permeation chromatography. The chromatographic properties of the 150 kDa antigens were unaffected by reduction using dithiothreitol, but incubation in acetate buffer, pH 3.0, dissociated the 150 kDa complex into at least two components with molecular weights of 70 to 100 kDa and 20 to 40 kDa. Only the smaller component displayed TSTA activity. The presence of two major components in the 150-kDa antigen was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TSTA activity was sensitive to digestion with pronase, papain, chymotrypsin, and alpha-mannosidase, but resistant to DNase, RNase, neuraminidase, trypsin, endoglycosidase H, and a mixed-function glycosidase. In addition, the TSTA activity was unaffected by heating. These data demonstrate that MCA carcinogenesis results in the expression of immunologically unique epitopes on biochemically related glycoproteins and suggest a unified mechanism for the generation of TSTA polymorphism.

Extraction of a murine tumor-specific antigen from cells and plasma membranes using 1-butanol: augmentation of antigen yield by colchicine.

The purpose of this investigation was to examine the ability of single-phase aqueous solutions of 1-butanol to release immunoprotective tumor antigen activity from partially purified plasma membranes of the methylcholanthrene-induced fibrosarcoma, MCA-F. Tumor antigen activity was assessed by s.c. immunization of syngeneic C3H/HeJ mice 10 days prior to supralethal challenge. Although brief incubation of intact MCA-F cells in 2.5% butanol releases potent immunoprotective activity, application of this protocol to plasma membranes did not result in antigen extraction. Modification of the extraction protocol using higher concentrations of butanol and longer extraction times did release measurable tumor antigen activity. However, a significant amount of the membrane-associated activity remained with the insoluble membrane fraction, as demonstrated by the immunoprotective capacity of the extracted membranes. The dramatic difference in the extractability of antigen from intact cells and plasma membranes suggested that membrane architecture may influence antigen release. To investigate this possibility, we extracted with butanol MCA-F cells that had been preincubated in colchicine. Treatment of cells with colchicine significantly potentiated the extraction of tumor antigen activity. Augmentation of antigen yield was also observed when plasma membranes were pretreated with colchicine prior to 2.5% butanol extraction. These results suggest that the tumor-specific transplantation antigen may be directly or indirectly associated with the cytoskeleton underlying the plasma membrane.