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Objective To determine whether genes participating in programmed cell death, including bcl-2, IL-10, Fas-L, and CTLA-4, may contribute to the genetic predisposition to systemic lupus erythematosus (SLE). Methods First, intragenic markers for the bcl-2, IL-10, Fas-L, and CTLA-4 genes were characterized and their extent of polymorphism in normal populations was determined. The allelic distribution of these gene markers in a large Mexican American SLE cohort of 158 patients and 223 ethnically matched controls was determined using fluorescent-labeled primers and semiautomated genotyping. Results The bcl-2, Fas-L, and IL-10 loci showed significantly different allelic distribution in SLE patients compared with controls, indicating an association between these genes and SLE. No association was found between SLE and the CTLA-4 gene. Further analysis revealed a synergistic effect between susceptibility alleles of the bcl-2 and IL-10 genes in determining disease susceptibility. Alone, the presence of each of these alleles was associated with a moderate increase in SLE risk, while the occurrence of these alleles together increased the odds of developing SLE by more than 40-fold. Conclusion The results suggest that individuals carrying specific genotypes of both bcl-2 and IL-10 are at significant risk of developing SLE.

BACKGROUND Several studies have recently suggested that the immune response to malignant growths is regulated by distinct patterns of type 2 cytokine production. These cytokines, regulating the cytotoxic T-lymphocyte response in patients with advanced cancers, may be associated with disease progression. Evidence suggests that the T Helper 1 (TH1) and T Helper 2 (TH2) types of reaction are reciprocally regulated in vivo. The immunomodulator AS101 (ammonium trichloro[dioxoethylene-O,O']tellurate) was found to stimulate mouse and human cells to proliferate and secrete a variety of cytokines. Clinical trials using AS101 on cancer patients are now in progress. PURPOSE The aim of this study was to evaluate the ability of AS101 to modulate TH1 and TH2 responses in tumor-bearing mice and in patients with advanced cancer. In addition, we investigated the association between the predominance of each type of response with the antitumoral effects of AS101. METHODS Mice into which Lewis lung carcinoma (3LL) had been transplanted (n = 221) and cancer patients (n = 13) were treated with AS101 on alternate days, at 10 micrograms/mouse intraperitoneally, or for the patients, at 3 mg/m2 intravenously. The types were sarcoma, melanoma, and colon, lung, ovarian, and renal cancers. Cytokine levels were determined by immunoassay kits and compared with the paired Student's t test: in mice, they were tested in spleen cell supernatants; in humans, in sera and mononuclear cell supernatants. The chi-squared test was used to compare tumor volumes. All P values represent two-sided tests of statistical significance. RESULTS Our results show that treatment of mice and patients with AS101 results in a clear predominance in TH1 responses, with a concomitant decrease in the TH2-type response. This was reflected by a significant enhancement in interleukin 2 (IL-2) and interferon gamma (IFN gamma) levels (P < .01) paralleled by a substantial decrease in IL-4 and IL-10 (P < .01). Moreover, the concentration of IL-12 was significantly increased (P < .01) in AS101-treated patients who also showed enhanced levels of natural and lymphokine-activated killer cell-mediated cytotoxicity. The statistically significant increases in IL-2 and IFN gamma levels, paralleled by the pronounced decrease in IL-4 and IL-10 in the AS101-treated mice, were associated with its antitumoral effects. In addition, systemic cotreatment of 3LL-transplanted mice with AS101 and anti-IL-12 antibodies partly abrogated the antitumoral effect of AS101. CONCLUSIONS Immunotherapy with AS101 enhances TH1 function while interfering with the TH2 response. This TH1 trend may be related to the antitumor effects of AS101. IMPLICATIONS Isolation and characterization of a distinct cytokine pattern in patients with advanced cancer treated with AS101 may contribute to the development of intervention strategies using this compound.

In several studies, the anabolic hormones insulin-like growth factor-1 (IGF-1) and insulin attenuated several metabolic changes associated with cancer cachexia. In the present study, we evaluated the effect of these hormones on the cachexia associated with colon-26 (C-26) tumor. Healthy age-matched and tumor-bearing mice were treated with two daily doses of IGF-1 (50 micrograms/kg in toto), or insulin (1 U in toto). Determinants of cachexia were body and tumor weight, epididymal fat pad and serum glucose concentrations. Neither IGF-1 nor insulin treatment had a significant effect on the cachectic parameters of C-26-bearing mice. These hormones were biologically active, being capable of inducing weight gain in hypophysectomized mice and hypoglycemia, respectively. Although IGF-1 and insulin have been used to treat cancer-related weight loss, the research presented here suggests that the beneficial effect of these hormones is not universal.

The immunomodulator ammonium trichloro(dioxyethylene-0–03′)tellurate (AS101) has previously been found by us to have radioprotective properties when injected into mice before sublethal and lethal doses of irradiation. AS101 also was found to protect mice from hematopoietic damage caused by various chemotherapeutic drugs. Based on these findings, phase II clinical trials with cancer patients treated with AS101, in combination with chemotherapy, are currently underway. In the present study, we wanted to assess the role of several cytokines in the radioprotection conferred by AS101. We show that the administration of neutralizing antibodies against interleukin-1 (IL-1) receptor, IL-6 receptor, IL-6, tumor necrosis factor (TNF), or stem cell factor (SCF) completely abrogates the ability of AS101 to increase the survival of lethally irradiated mice. Moreover, the injection of each of these antibodies reduces the ability of AS101 to increase the number of BM, spleen cells, and the number of circulating neutrophils, lymphocytes, and platelets in irradiated mice. In addition, these antibodies abrogate the enhancing effect of AS101 on the secretion of IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor, all of which decrease significantly in sublethally irradiated mice. By contrast, the injection of anti-IL-2 receptor antibody or control Igs to AS101- treated mice does not interfere with the radioprotective effects of the compound. These results suggest a role for IL-1, IL-6, TNF alpha, and SCF in the radioprotective effect of AS101. Because cytokine toxicity remains a significant concern, the clinical application of AS101, which has no toxicity, is particularly valuable.

Among other effects, prostaglandins (PG) of the E series are known to inhibit several acute and chronic inflammatory conditions in vivo and proinflammatory cytokine production by activated macrophages in culture. The research presented here demonstrates that the inhibitory effect of PGE2 on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages involves IL-10. In a dose-dependent manner, PGE2 inhibits LPS-induced release of TNF-alpha and IL-6, but not of lactate or nitric oxide. The decrease in the level of these cytokines is inversely proportional to the increase in immunoreactive IL-10. This differential inhibitory effect of PGE2 is mimicked by agents that elevate intracellular levels of cAMP, but not cGMP. Neutralizing anti IL-10 antibody but not neutralizing antibodies against other macrophage secretory products (IL-6, leukemia inhibitory factor, and transforming growth factor beta [TGF-beta]), significantly reverse the potent inhibitory effect of PGE2. In vivo, the administration of PGE2 before LPS challenge significantly reduces circulating TNF-alpha and IL-6 levels. Anti-IL-10 antibody substantially enhanced the LPS-induced TNF-alpha and IL-6 levels in mice that received either LPS alone or LPS plus PGE2. These results suggest that the anti-inflammatory effect of PGE2 on mononuclear phagocytes is mediated in part by an autocrine feedback mechanism involving IL-10.

This report demonstrates the ability of the anti-cancer drug suramin to interfere with the binding of interleukin (IL)-1 to its receptor and to inhibit IL-1-induced biological activities. In a radioreceptor cell based assay, suramin inhibits the binding of IL-1 alpha to several murine cell lines expressing predominantly type I and type II IL-1 receptors. Affinity cross-linking experiments using IL-1 alpha and EL-4.6.1 cells confirms that suramin inhibits the binding of the ligand to the 80 kDa IL-1 type I receptor. In contrast, suramin fails to displace significantly prebound IL-1. In a cell-free system, suramin prevents the binding of IL-1 alpha and IL-1 beta to murine and human recombinant soluble type I IL-1 receptors. For example, the IC50 for suramin inhibiting IL-1 alpha and IL-1 beta binding to soluble human IL-1 receptor were 204 microM and 186 microM, respectively. The suramin analogues, NF-058 and NF-103 (which bear the same number of sulfate groups as suramin), are between three- and ten-fold less active than suramin in inhibiting IL-1 binding to EL-4.6.1 cells, and to recombinant soluble IL-1 receptor. Furthermore, in a dose-dependent manner suramin prevents several IL-1 mediated biological responses, including thymocyte proliferation, PGE-2 synthesis and IL-6 production. The inhibitory effect of the drug can be significantly reversed by the addition of excess cytokine. Taken together, the results indicate that suramin is a competitive IL-1 receptor antagonist. Because IL-1 participates in a broad range of immunological and inflammatory functions, the data suggest that suramin administration may influence important activities beyond those associated strictly with tumor inhibition.

Reports from several laboratories have suggested that interleukin 6 (IL-6) may play a role in the process of bone resorption. We have extended these studies by examining the role of IL-6 in fetal rat long bone (FRLB) resorption stimulated by a variety of agents, including parathyroid hormone (PTH); 1,25 dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ); interleukin 1 (IL-1); tumour necrosis factor alpha (TNF-α) and lipopolysaccharide (LPS). This model of bone resorption does not require the generation of osteoclasts in order to elicit a resorptive response and allowed us to assess whether IL-6 can directly affect osteoclastic bone resorption. We confirmed previous studies which showed that exogenous recombinant murine or human IL-6 does not stimulate bone resorption and demonstrated that IL-6, when added prior to the addition of parathyroid hormone, caused a significant but somewhat variable inhibition at 120 hours. Exogenous PGE 2 stimulated both IL-6 production and resorption in FRLB cultures in a concentration-dependent manner. Endogenous production of IL-6 in fetal rat long bone (FRLB) cultures was stimulus dependent and generally correlated with prostaglandin E 2 (PGE 2 ) levels in the same cultures. However, endogenous IL-6 production did not correlate with the extent of bone resorption, except when IL-1 and PGE 2 were used as stimuli. Addition of indomethacin and diclofenac to IL-1 stimulated cultures demonstrated that both the IL-6 production and bone resorption were largely PGE-2 dependent. Neutralizing anti-IL-6 antibodies inhibited IL-6 activity in FRLB cultures but did not affect bone resorption, even in the IL-1 stimulated cultures. Taken together, these data suggest that, in general, IL-6 produced by fetal rat long bones correlates with prostaglandin production and is not directly related to resorption in FRLB cultures, irrespective of the stimulus employed. However this does not exclude the possibility that IL-6 may be involved in the maturation or differentiation of osteoclasts.

Neoplastic diseases are frequently associated with metabolic changes collectively known as cancer cachexia. The presence of cachexia complicates therapeutic intervention and is an important cause of death in cancer patients. At present there is no effective treatment for cachexia. Recently, the involvement of interleukin-6 (IL-6) in the wasting of colon-26 adenocarcinoma-bearing mice was demonstrated. The research presented here establishes an anticachectic role for the experimental drug suramin, since it partially blocks (up to 60%) the catabolic effects associated with the growth of this tumor in vivo. Suramin prevents the binding of IL-6 to its cell surface receptor subunits, as demonstrated by radioreceptor binding assay and affinity crosslinking experiments. Furthermore, the uptake of radioactive IL-6 by the liver is significantly reduced in suramin-treated mice. On the other hand, the drug is approximately 10-fold less potent in inhibiting the binding of tumor necrosis factor-alpha to indicator cell line in vitro and fails to block liver uptake of this cytokine in vivo. Collectively, these results suggest that suramin inhibits cancer-associated wasting, in part by interfering with the binding of IL-6 to its receptor. Whether suramin inhibits the action of other factors/cytokines that may also participate in colon-26-mediated cachexia is not yet known.

The precise mechanisms responsible for increased calcium levels in patients with cancer are not fully understood. In a recent study, the participation of interleukin (IL)-6 as an important mediator of key parameters of cancer cachexia in the colon-26 adenocarcinoma was reported. Here, we show that in addition to cachexia, C-26 tumour bearing mice also develop hypercalcemia. Treatment of these mice with 5′ deoxyfluorouridine significantly reduces tumour size and inhibits both hypercalcemia, cachexia, and elevated serum IL-6. Moreover, monoclonal antibody to mouse IL-6 prevents both the cachexia and the hypercalcemia and reduces serum IL-6 levels in C-26 tumour bearing hosts. The administration of a bisphosphonate compound (Clodronate) reverses the hypercalcemia but has no effect on tumour burden, serum IL-6 levels, or wasting. We conclude that tumour-derived IL-6 plays a role in the pathogenesis of the C-26 associated hypercalcemia, and that the increase of serum calcium does not by itself mediate cachexia.

In the present report, we show that progressive growth of the immunogenic C57BL/6J sarcoma, MCA/76-9, was accompanied by an increase in serum interleukin-6 (IL-6) activity. The possible pathways leading to the induction of IL-6 release by the tumor cells are described. It was shown that macrophage products IL-1 alpha, IL-1 beta, and to a lesser extent, TNF alpha, induced the tumor cells in vitro to transcribe the IL-6 gene and release the gene product. IL-1 induced significantly more IL-6 mRNA and bioactivity than TNF alpha, although both cytokines induced a cumulative increase of bioactivity in the supernates over a period of 24 h. The tumor cells were shown to express receptors for IL-1 alpha, which could be blocked with anti-IL-1 receptor antibody. Given the previous reports that tumor-associated macrophages expressed both IL-1 alpha/beta and TNF alpha, the data suggest, first, that the mutual interaction of tumor cells and macrophages in situ may contribute to the observed increase in circulating IL-6 activity, and second, that the release of IL-6 in vivo may serve to regulate both anti-tumor immune responses and suppressor mechanisms.

In this report we describe an experimental model of cachexia that fulfills the criteria of an early effect with a small tumor mass not related to the growth rate of the tumor, and progressive wasting of muscle and fat without a detectable loss of appetite. C-26.IVX is a cell line derived from murine colon-26 adenocarcinoma which retains the transplantability of the original tumor and induces true cachexia in syngeneic hosts. Evidence is presented to support a role for interleukin (IL-6) as a cachectic factor in the development of cancer cachexia in this model system. Thus, increasing levels of IL-6 in C-26.IVX-bearing mice correlate with the development of cachexia. If the primary tumors were resected, mice gained weight and the levels of IL-6 in the serum were reduced significantly. Moreover, monoclonal antibody to murine IL-6 (but not anti-tumor necrosis factor antibody) was able to significantly suppress the development of key parameters of cachexia in tumor bearing mice.

The ceramide analogue 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP) (particularly the D-threo isomer, D-PDMP) caused inhibition of cell growth in some types of cells, and this growth-inhibitory effect has been attributed to inhibition of UDP-Glc:Cer beta-Glc transferase, resulting in reduced glycolipid synthesis and increased free ceramide [Inokuch, J., & Radin, N. S. (1987) J. Lipid Res. 28, 565-571; Okada, Y., et al. (1988) FEBS Lett. 235, 25-29]. In view of increasing evidence that the T cell proliferative immune response is modulated by glycosphingolipids (GSLs), the reagent D-PDMP was used to evaluate the role of GSLs in this respect. Con A induced or PHA-induced mitogenesis of C3H/HeJ mouse splenocytes, as well as IL2-dependent CTLL cell growth, were strongly inhibited in a dose-dependent manner when cells were preincubated in the presence of 5-10 microM D-PDMP, but not with its stereoisomer L-PDMP. Closely associated with this growth-inhibitory effect in the presence of D-PDMP, levels of essentially all GSLs, including GM3 and other gangliosides, were greatly reduced, whereas ceramide accumulated. Importantly, metabolically labeled radioactive bands, corresponding to free sphingosine and N-monomethylsphingosine, were found to be present in very small quantities (5-12%) relative to the band corresponding to N,N-dimethylsphingosine (DMS), which showed significant accumulation in D-PDMP-treated lymphocytes. The quantity of IL2 receptors and their affinity to IL2 on T cells did not change, but IL2-dependent tyrosine phosphorylation was greatly stimulated, following D-PDMP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined the effect of polymorphonuclear cells on the release of tumor necrosis factor (TNFalpha) in endotoxin-treated macrophages. Human peripheral blood neutrophils were co-cultured with mouse peritoneal macrophages stimulated with lipopolysaccharide (LPS). In a dose-dependent manner, FMLP (n-formyl-methionyl-leucyl-phenylalanine) augmented the release of TNFalpha by LPS-stimulated macrophages in the presence, but not in the absence, of neutrophils. The stimulating effect of neutrophils on macrophages was reversed by catalase, suggesting that the release of hydrogen peroxide from neutrophils was responsible for augmenting macrophage TNFalpha. Moreover, the direct addition of hydrogen peroxide to macrophages resulted in an increased secretion of TNFalpha. In addition, insertion of a porous membrane between the neutrophils and macrophages cancelled the effect, indicating that adherence of neutrophils may be necessary for augmentation of TNFalpha release. In summary, the data suggest that hydrogen peroxide released from stimulated neutrophils may act as an activator of macrophage function by increasing their release of TNFalpha.

The immunomodulator AS101 has been previously shown to confer protection upon BALB/c mice infected with the intraerythrocytic parasite Babesia rodhaini (B. rodhaini). The present study focuses on the effect of AS101 administration on the acute phase of babesial infection where T helper cell subset patterns-TH1/TH2-were assessed in heavily infected mice. Secretion of cytokines of the TH1 subset (IL-2, IFN-gamma, IL-12) and of the TH2 subset (IL-10, IL-4) as well as TGF-beta was measured following the administration of AS101 2 weeks before parasite infection. Our results demonstrate that the parasites suppress IL-2 protein and IL-12 mRNA and that AS101 upregulates their secretion: IL-2, 8 u/ml vs 34 u/ml, respectively; IFN-gamma protein, 2370 pg/ml vs 4777 pg/ml, respectively. Conversely, babesial infection results in the upregulation of IL-10 and IL-4 proteins and TGF-beta transcripts, whereas AS101 downregulates their production: IL-10, 1800 pg/ml vs 360 pg/ml, respectively; IL-4, 58.3 pg/ml vs 24.5 pg/ml, respectively. A possible escape mechanism induced by B. rodhaini is suggested, starting with IL-10 inhibition of macrophage activities leading to a suppression of the TH1 response and of IL-2 in particular. It is therefore possible that AS101 may protect infected mice by activating cellular-mediated immunity and concurrently balancing the TH subset responses. It is suggested that AS101 may be effective as an antiparasitic drug.

AS-101 is a tellurium-based compound with known immunomodulating properties. The ability of AS-101 to potentiate the effects of chemotherapeutic drugs and augment cytokine production in vivo has led to clinical trials on AS-101 which are currently being carried out in cancer patients. In the present study we show that AS-101 selectively augments the release of TNF alpha and IL-1 alpha and inhibits the release of IL-10 by lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and human monocytes. It does not significantly affect the release of IL-6 or leukemia inhibitory factor (LIF). By itself AS-101 does not induce the release of any of these cytokines. Analysis of IL-10 and TNF alpha RNA levels using semiquantitative PCR reveals that AS-101 blocks the transcription of IL-10 mRNA, but does not significantly affect TNF alpha mRNA. Although both AS-101 and interferon (IFN)-gamma inhibit IL-10, AS-101, unlike IFN-gamma, does not prime macrophages for LPS-induced nitric oxide release and does not appear to significantly affect monocyte HLA-DR expression. Our data suggest that AS-101 is a partial IFN-gamma agonist and may explain the shift toward the release of Th-1 type cytokines observed in AS-101-treated patients.

Previous studies have shown that the administration of concanavalin A (ConA) into mice induces immune-mediated liver injury, which can be largely abrogated by neutralizing tumor necrosis factor(TNF)alpha. Vesnarinone is an experimental drug which is known to inhibit TNF alpha release. Here we demonstrate that vesnarinone inhibits ConA-induced hepatic injury. In a dose-dependent manner, vesnarinone inhibits in several mouse strains the increase of serum aminotransferase concentrations. additional experiments show that vesnarinone inhibits ConA-mediated accumulation of DNA fragmentation in the liver. Furthermore, the drug significantly reduces the levels of circulating TNF alpha and interleukin-6 (IL-6). Vesnarinone does not modulate TNF alpha and IL-6 action on hepatic cells, as shown by its failure to reduce the cytokine specific-stimulation of acute phase plasma proteins in the rat hepatoma H-35 cell line. Neither vesnarinone nor anti-TNF alpha protect against direct liver injury induced by a sublethal dose of agonist anti-Fas (CD95) antibody. Taken together, these results suggest that vesnarinone blocks hepatic injury, in part by inhibiting the release of TNF alpha in vivo.

Several recent reports presented conflicting data on the action of IL-4 and IL-13 in regulating the release of proinflammatory cytokines by human monocytes. Here we show that the regulation of cytokine release by IL-4 and IL-13 could be either inhibitory or stimulatory in LPS-treated murine peritoneal macrophages. When macrophages were treated with IL-13 or IL-4, between 6 and 24 hr prior to endotoxin challenge, TNF alpha and IL-6 levels were significantly augmented. On the other hand, when the cells were cotreated with LPS plus IL-13 or IL-4, the release of TNF alpha and IL-6 was inhibited. These effects of IL-4 and IL-13 were associated with the modulation of IL-10; pretreatment resulted in a decrease, whereas cotreatment gave rise to a dramatic increase in IL-10 levels. The inhibitory effect of IL-4 and IL-13 on the release of TNF alpha was partially reversed by neutralizing anti-IL10 antibody, and the inhibition of IL-6 release was completely reversed by the antibody. These data suggest that the mechanism of action of IL-13 and IL-4 in modulating macrophage TNF alpha and IL-6 release partially involves IL-10.

Vesnarinone is an experimental drug that has been used successfully in the treatment of congestive heart failure patients. In this report we investigate the effect of vesnarinone on the cytokine secretory products of mononuclear phagocytes. In a concentration-dependent manner, the drug inhibits the endotoxin(LPS)-stimulated release of tumor necrosis factor (TNF) alpha and suppresses interleukin(IL)-6 release, but does not affect the release of IL-1 alpha, IL-10 and leukemia inhibitory factor (LIF) by mouse peritoneal macrophages. Using competitive polymerase chain reaction (PCR) analyses, we find that vesnarinone significantly reduces TNF(alpha), but not IL-10 mRNA. In addition to LPS, the drug inhibits TNF(alpha) release induced by several other stimuli. The inhibitory effect of the drug on the TNF(alpha) biosynthesis can be observed in differentiated human monocytes, in macrophage cell lines, and in synovial adherent cells from rheumatoid arthritis patients. Although the precise mode of action of vesnarinone in the signal transduction pathway leading to the selective inhibition of TNF(alpha) is not known, the drug might be useful in the treatment of diseases involving that cytokine.

The experimental drug suramin has been shown to possess several immunosuppressive properties. In this study we investigated the effect of suramin on the development of experimental autoimmune uveoretinitis (EAU) in mice and in rats. EAU was induced either by active immunization with a uveitogenic protein or peptide, or by the adoptive transfer of a uveitogenic T cell line. The development of EAU was assessed by clinical evaluation as well as by histopathology. Immunological responses were measured by delayed type hypersensitivity (DTH), lymphocyte proliferation, and serum antibody levels to the immunizing antigen. Suramin treatment was most effective in suppressing EAU when started concurrently with immunization (afferent). Treatment was less effective in suppressing disease when first administered 7 days after immunization or when given to animals that received an adoptive transfer of uveitogenic T cells (efferent). The effect of suramin on DTH and lymphocyte proliferation roughly paralleled its effect on EAU. Aferent treatment of mice with suramin completely suppressed anti-IRBP antibody titers. Interestingly, animals receiving efferent treatment had unreduced IgM levels but little or no IgG, suggesting prevention of the IgM-to-IgG switch. Depressed in vitro lymphocyte proliferative responses in animals treated with suramin during the afferent stage suggested that the suppressive effect on disease was due at least in part to an inhibition of antigen priming. Our results suggest that suramin merits further investigation as a potential treatment for some types of uveitis.

In this study, the ability of recombinant human macrophage (M) and murine granulocyte-macrophage macrophage (GM) colony stimulating factor (CSF) to affect both basal and stimulated bone resorption in fetal rat long-bone organ cultures was assessed. It was found that M-CSF does not affect basal bone resorption or bone resorption stimulated by parathyroid hormone, recombinant human interleukin 1β, prostaglandin E 2 (PGE 2 ), and 1,25 dihydroxy vitamin D 3 . Specifically, M-CSF at concentrations as high as 30 nM (1 μg/mL) did not modulate 45 Ca release from fetal rat long bones stimulated by these agents. The addition of recombinant murine GM-CSF (at equal molar concentration to M-CSF) also did not affect bone resorption stimulated by parathyroid hormone and interleukin 1β. On the other hand, GM-CSF stimulated basal bone resorption over a 120-h period and augmented the resorption mediated by exogenous PGE 2 over a 48-h incubation. In addition, GM-CSF was shown to stimulate production of endogenous PGE 2 in cultures of bone rudiments. These effects on bone resorption were blocked by the addition of prostaglandin synthesis inhibitors and specific antibodies to murine GM-CSF. These data indicate that M-CSF does not act as a regulator of bone turnover, but GM-CSF may cause bone resorption by stimulating the synthesis of PGE 2 in bone.

Delayed-type hypersensitivity (DTH) responses served in this study as an experimental model for the analysis of genetic regulations of T-cell responses. Educated irradiated cells from H-2b mice mediated responses in syngeneic recipients, whereas mice of the a, d, f, k, and s haplotypes were nonresponders to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys)[(T,G)-A--L]. These results suggest that cell-mediated immune responsiveness to (T,G)-A--L is linked to the H-2 complex, as was shown for humoral responses. Educated irradiated T cells of F1 hybrids between high and low responders mediated DTH responses, which indicates that the gene(s) controlling the DTH responses is dominant. To analyze the genetic defect in DTH responses to (T,G)-A--L, we separated the T-cell activation phase from the effector phase that was determined in recipient mice. Two types of nonresponders were observed: (a) When lymphocytes of the a or k haplotypes were educated in a syngeneic environment and then transferred into hybrids between the parental (nonresponder x responder) F1 recipients, DTH responses could have been manifested. (b) On the other hand, no DTH responses could be mediated by transferring educated cells of the H-2s or H-2f origin into the appropriate F1 recipients. In addition, irradiated F1 cells that had been activated to (T,G)-A--L could not mediate DTH responses in both types of nonresponder recipients. These results suggest that T cells of H-2k or H-2a mice can be activated to generate DTH responses to (T,G)-A--L and that the defect in these mouse strains is expressed in another cell population needed for the manifestation of the DTH reaction in the recipient mice. In contrast, T cells of H-2s and H-2f origin cannot be activated to (T,G)-A--L and, thus, fail to manifest DTH responses.

The intercellular interactions and the site of the genetic defect in delayed-type hypersensitivity (DTH) response to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys) [(T,G)-A--L] has been studied in a system where the T-cell education phase was separated from the efferent phase. In the cellular response, T-T-cell collaboration is required, because T cell-depleted mice were unable to manifest DTH responses after they were transferred with educated and irradiated T cells. Reconstitution of adult thymectomized mice that were irradiated and supplemented with bone marrow cells after treatment with anti-Thy-1.2 serum and complement, with T cells but not with accessory cells gave rise to significant responses. Educated, radioresistant cells required the presence of normal radiosensitive T cells for successful DTH responses to (T,G)-A--L. The genetic defect of nonresponder H-2k and H-2a mice has been located in the above-mentioned, second T-cell population that participates in the efferent phase of this immune reaction. Further characterization revealed that the educated cells are of the Lyt1+ phenotype and that the second normal T cells are expressing the Lyt 1+,2+,3+ phenotype. Thus, the genetic defect of H-2k and H-2a mice in the DTH response to (T,G)-A--L is expressed on the non-antigen-stimulated Lyt 1+,2+,3+ T cells.

The crossreactivity between the random synthetic polypeptide antigen poly(Tyr,Glu)-poly(DLAla)--poly(Lys) [(T,G)-A--L] and its ordered-sequence analogs (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys) [(T-T-G-G)-A--L] and (Tyr-Glu-Tyr-Glu)-poly(DLAla)--poly(Lys) [(T-G-T-G)-A--L] at the level of humoral and cellular responses was studied. For delayed type hypersensitivity responses, (T,G)-A--L-activated T cells could be challenged with the homologous antigen as well as with the ordered analogs. T cells activated by (T-T-G-G)-A--L could be challenged with either the homologous antigen or with (T,G)-A--L but not with (T-G-T-G)-A--L. Similarly, no cross stimulation was observed between (T-G-T-G)-A--L-activated cells and (T-T-G-G)-A--L, whereas (T,G)-A--L could challenge the latter cells to mediate significant responses. Similar but not identical cross reactions were observed when primed spleen cells or lymph nodes were transferred to irradiated recipients that were boosted for the production of antibodies. In contrast to observations at the level of cellular responses, (T-G-T-G)-A--L-primed spleen or lymph node cells could not be boosted with (T,G)-A--L for the production of detectable amounts of antibodies, although boosting with the homologous antigen resulted in significant levels of (T-G-T-G)-A--L-specific antibodies. Transfer experiments in which mixtures of T and B cells, each primed to a different ordered polypeptide antigen, were injected into irradiated recipients showed that successful cooperation occurs provided that the boost is given with the T-cell-specific antigen. The antibodies produced were specific to the antigen used for B-cell priming. The T-cell-B-cell collaboration probably occurs through specific determinants that are shared between the two antigens in which the ordered peptides are attached to the same multichain polymer and that are recognized by both the T and the B cells.

We previously demonstrated that although human cytotoxic T lymphocytes (CTL) generated against a lymphoblastoid cell line (LCL) mutant that had lost DR and DQ (formerly known as MB) expression were directed primarily at class I antigens, the lytic activity of such CTL could be inhibited by monoclonal antibodies (MoAb) against monomorphic determinants on DR molecules. Furthermore, the inhibitory effect was found to occur by binding of the MoAb to the target cell, not the effector cell, because lysis of LCL target cells not expressing DR was not inhibited. In this paper we extend this phenomenon to show (i) that MoAbs directed against DQ and DP class II gene products also inhibit the lysis of LCL target cells by CTLs recognizing class I antigens; and (ii) that not all DR-specific MoAbs blocked target cell lysis by non-DR specific CTLs. In addition, when PBLs were used to stimulate the generation of CTLs in mixed leukocyte culture (MLC), inhibition using anti-class II (DR, DQ, and DP) MoAb occurred only when LCLs but not PHA blasts were used as targets. This might be explained by elevated expression of class II molecules on LCLs compared to PHA blasts.

The effect of transforming growth factor type beta 1 (TGF-beta 1) on mononuclear phagocytes (macrophages), cells which play an important role in the inflammatory response resulting from tissue wounding, was investigated. We found that fibronectin production by murine inflammatory macrophages is significantly enhanced by highly purified human TGF-beta 1 in a time- and dose-dependent manner. Specifically, 2 pM TGF-beta 1 was sufficient to cause significant elevation of fibronectin levels, which peaked between 24 and 48 hr of incubation. Both TGF-beta 1-induced and basal levels of fibronectin were completely abolished by cycloheximide, suggesting that protein synthesis was required. Furthermore, fibronectin mRNA levels were significantly enhanced in TGF-beta 1-treated macrophages. The inductive capacity of TGF-beta 1 appeared specific since other agents such as phorbol myristate acetate and endotoxin failed to induce fibronectin production. Since macrophages have been recently shown to secrete an inactive form of TGF-beta 1, the ability of this precursor molecule to induce fibronectin production was tested. It was found that partially purified human platelet latent TGF-beta 1 could not induce fibronectin synthesis, whereas acid treatment of the same preparation which releases mature TGF-beta 1 enhanced fibronectin production. Taken together, results presented here suggest that inflammatory macrophages can directly contribute to the formation of extracellular matrix upon interaction with TGF-beta 1 and that these cells lack the ability to augment fibronectin production in response to a platelet-derived latent form of TGF-beta 1.

DTH responses were evaluated in different strains of mice shown to be resistant or sensitive to leukemogenesis by the radiation leukemia virus variants A-RadLV and D-RadLV. A significant response was observed only in the H-2 complex-linked resistant haplotypes to RadLV leukemogenesis. The DTH response could be transferred by immune cells of mice resistant to the appropriate RadLV variant. Thus, an inverse relationship between the leukemogenic activity of the virus and its immunization ability expressed by DTH response was demonstrated in different mouse strains.

We have developed an in vitro system for the activation of T cells in order to get a better insight into the genetic and molecular mechanisms involved in the generation of delayed-type hypersensitivity (DTH) effector T cells. Low doses of fowl γ-globulin (FγG) as well as the synthetic polypeptide (T,G)-A-L were bound to splenic adherent cells and served as immunogens for the in vitro sensitization of lymphocytes. In parallel, (T,G)-A-L-specific T cells were activated in vivo in irradiated recipient mice. The ability of the in vitro - and in vivo -activated cells to mediate DTH responses was determined in naive recipient mice by the radioisotopic ear assay. Twenty to thirty × 10 6 “educated” cells were sufficient to elicit significant DTH responses. Irradiation of the spleen cells prior to their transfer resulted in higher responses. The DTH reactivity was transferable by nylon wool-enriched T cells but not by a Thy 1.2-depleted population indicating the T-cell dependency of the response. The in vitro and in vivo antigen-activated T-cell population exhibited also helper-cell activity as determined by their cooperation with B cells in adoptive transfer experiments.

We have utilized cloned T cell reagents and ionizing radiation-induced mutants of an HLA heterozygous lymphoblastoid cell line (LCL) to investigate the determinants detected by the cell-mediated lympholysis (CML) assay. Cells of an LCL clone, 721.501, and HLA haplotype loss mutant expressing the HLA-A2-Cw1-Bw51-DR1-Dw1-DQw1-DPw2-GLO haplotype were used as sensitizing cells for responder cells in vitro. “Cloned” reagents were generated by single-cell deposition of cells of a bulk reagent primed against 721.501 cells. Those clones were screened for cytolytic activity against HLA loss mutant targets (derived from LCL 721) of four different categories; HLA-A2 loss only. A2-Cw1-Bw51 loss, A2-Cw1-Bw51-DR1-DQw1 loss, and the entire HLA haplotype loss. Of 196 clones tested, 36 were cytolytic, including three anti-A2, five anti-Bw51/Cw1, 12 anti-DR1/DQw1, 13 anti-DP region associated with DPw2, and three of undetermined specificity, based on cytolytic patterns against the HLA loss mutant targets. Of 25 anti-HLA class II lytic clones, 23 (92%) fitted the cahracteristics of helper cell-independent cytolytic T cells (HITc), whereas only two of eight (25%) anti-class I clones were HITc. The 13 anti-DP region clones were divided into three subgroups defined by blocking by anti-FA and not Tu39 monoclonal antibodies (MoAb), by Tu39 and not anti-FA, and by both MoAbs.

Monoclonal antibodies directed at monomorphic determinants on A,B,C (w6/32) or DR (L243, S4/24, S8/8) HLA antigens were used to inhibit the cytotoxic activity of class I- or class II-reactive CTL clones. Anti-class I (HLA-Bw62) cytotoxic T lymphocyte CTL clones were inhibited by w6/32, but not by L243, when tested on PHA blasts and LCL as targets. Interestingly, S4/24 and S8/8 demonstrated a differential blocking ability of class I reactive clones; these two monoclonal antibodies could not block Bw62-directed CTL clones when using PHA blasts as targets but strongly inhibited the cytotoxic activity of the same clones on LCLs as targets.

IFN-gamma can induce the expression of both class II histocompatibility antigens (Ia) and the lymphocyte function associated (LFA)-1 antigen on murine peritoneal macrophages. We have examined the molecular changes which lead to altered expression of these two cell surface proteins to determine whether they are regulated by similar or independent mechanisms. While I-A antigen expression can be induced or enhanced by treatment of macrophages with either phorbol diesters and/or the Ca2+ ionophore A23187, these agents had no effect upon expression of LFA-1 under similar conditions. Macrophages from the A/J strain mouse exhibit a deficiency in their sensitivity to IFN-gamma which is seen in our studies as an inability of IFN-gamma to elevate I-A antigen expression. However, expression of I-A could be modulated in these cells by treatment with either phorbol diesters or A23187. In contrast, IFN-gamma could induce LFA-1 antigen on A/J derived macrophages and this was not affected by either phorbol or A23187. Thus these two antigens, despite coordinate expression in response to IFN-gamma in normal mouse strains, are clearly regulated independently. These results suggest that IFN-gamma generates at least two independent molecular events in macrophages which ultimately modulate the expression of cell surface proteins important to the performance of activated functions.

Following secondary stimulation of mixed leucocyte culture (MLC) populations with a class I plus II HLA difference, antigen-specific suppressor-effector cells of the OKT8 + 4 − phenotype are generated. On the other hand, stimulation with a class II HLA genetic difference only gave rise to OKT4 + suppressor T cells. These effector cell populations which were maintained as “lines” by the addition of exogenous T cell growth factor (TCGF) and feeder cells, mediated antigen-specific suppression over a 6–8 week period during which they were assayed. The data reported here demonstrate that the phenotype of suppressor cells, generated in allogeneic MLC, depends on the nature of the class of HLA antigen recognized as disparities in the suppressor-generating MLC. Moreover, both OKT8 + 4 − and OKT4 + suppressors, obtained following stimulation with class I plus II or class II genetic disparities, respectively, appear to be specific for DR (or related class II product) antigens.