Antibody-induced suppression and postsuppression stimulation of complement in vitro. II. Intracellular and extracellular changes in C4 during long-term C4 suppression in guinea pig splenic fragments

Suppression of the synthesis of the fourth component of complement in vitro was originally accomplished by exposing cultured guinea pig peritoneal cells to anti-C4 alloantisera. When guinea pig splenic fragments were used instead of peritoneal cells, equivalent antibody treatment produced C4 suppression of significantly longer duration, lasting weeks instead of days after removal of antibody. As with peritoneal cell monolayers, antibody treatment induced specific suppression of C4 followed by nonspecific stimulation of C4 and other proteins such as C2. Although IgG2 is more readily sequestered by splenic tissue, both IgG1 and IgG2 antibodies were effective in inducing and maintaining suppression. Experiments with radiolabeled antibody demonstrated that a small amount (less than 5%) of the original dose of antibody was retained by the splenic fragments. Because there was no continuous slow release of that antibody, long-term suppression of C4 cannot be explained as a fluid-phase neutralization reaction. Because antibody treatment might induce production of aberrant C4 molecules with no functional activity, C4 antigens was also studied. Tissue culture supernatants were assayed by using an ELISA for C4. In none of these experiments was extracellular C4 antigen detectable immediately after antibody treatment. Extracellular and intracellular C4 were immunoprecipitated from biosynthetically labeled tissue cultures and analyzed by SDS-PAGE. Antibody treatment suppressed intracellular C4 as well as extracellular C4. Although extracellular C4 levels of antibody-treated cultures eventually returned to levels comparable to untreated cultures, intracellular C4 levels of treated fragments remained lower than controls for the full period of observation (22 days). Therefore, a short (4-day) exposure to anti-C4 antibody induced long-term effects that profoundly altered regulation of C4 synthesis and secretion by cultured splenic macrophages.     

Heat shock temperature acclimation of normal secretory protein synthesis in barley aleurone cells

Exposure of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers to 40°C for a period of 3 h results in the selective suppression of the synthesis and secretion of hydrolytic enzymes; other normal cellular protein synthesis continues during heat shock. This suppression is correlated with secretory protein mRNA destabilization and the dissociation of stacked ER lamellae during heat shock (Belanger et al. 1986, Proceedings of the National Academy of Sciences USA 83, pp. 1354–1358). In this report we examined the effect of exposure to extended periods of heat shock. If exposure to 40°C was continued for a period of 18 h, the synthesis of α-amylase, the predominant secreted hydrolase, resumed. This was accompanied by increased α-amylase mRNA levels and the reformation of ER lamellae. Though initial exposure (3 h) to 40°C reduced protein secretion to ～10% of that observed in aleurone cells maintained at 25°C, exposure for prolonged periods (16–20 h) permitted the resumption of protein secretion to ～66% of non-heat-shocked control levels. The resumption of normal secretory protein synthesis during prolonged exposure to 40°C was correlated with an increase in the incorporation of [¹⁴C]glycerol into phosphatidylcholine and an increase in the ratio of saturated to unsaturated fatty acids in lipids isolated from ER membrane preparations. Increased fatty acid saturation has been demonstrated to enhance thermostability in biological membranes, and such changes in membrane composition may be important to the recovery of secretory protein synthesis at the ER.     

Recombinant rat IL-1beta and IL-6 synergistically enhance C3 mRNA levels and complement component C3 secretion by H-35 rat hepatoma cells

Hepatic synthesis of complement component C3 is regulated in part by inflammatory cytokines. Rat models are frequently employed to investigate pathogenic roles of complement and cytokines. However, cytokines obtained from species other than the rat were used in previous studies of cytokine regulation of C3 synthesis in rat hepatocytes or hepatoma cells. It is not known whether these prior reports predict hepatocellular responses evoked by rat cytokines. Therefore, H-35 rat hepatoma cells were employed to measure the effect of recombinant rat IL-1beta, IL-6, IFN-gamma, and TNF-alpha on C3 protein secretion and C3 mRNA levels quantified by ELISA and quantitative RT-PCR. Compared to untreated control cells, H-35 cells treated with IL-1beta, IL-6, and IFN-gamma increased C3 secretion approximately 10-, 4-, and 2-fold, respectively. TNF-alpha was toxic, precluding further analysis. IL-1beta and IL-6 demonstrated synergy with respect to the quantity and rate of increase of C3 mRNA measured and the magnitude of C3 protein secretion. Previous reports using non-rat cytokines did not consistently predict H-35 responses to rat cytokines. Consequently, we recommend the use of rat cytokines in rat models that include analysis of cytokine-mediated events.     

Urocortins and corticotropin releasing factor type 2 receptors in the hypothalamus and the cardiovascular system

In addition to urocortin (Ucn I), Ucn II and Ucn III were identified as endogenous ligands for corticotropin-releasing factor type 2 receptor (CRF2 receptor). CRF2 receptor is abundantly located in central hypothalamic ventromedial nucleus (VMH) and in peripheral cardiovascular system. In this mini-review, we focused on the roles of these urocortins and CRF2 receptor in the hypothalamus and the cardiovascular system. Ucn II mRNA was increased in the parvocellular part or the magnocellular part of the hypothalamic paraventricular nucleus (PVN) following immobilization stress or 3 days of water deprivation, respectively. Therefore, it is thought that Ucn II may modulate CRF and vasopressin synthesis in the PVN in a paracrine or autocrine fashion through PVN CRF2 receptor. The early and later phases of Ucn I-mediated feeding suppression may be CRF1 and CRF2 receptor-mediated events, respectively. Ucn II decreases food intake at a later phase, beyond 4 h post injection. A large dose of corticosterone increased plasma leptin and insulin levels as well as the levels of CRF2 receptor mRNA. Adrenalectomy, starvation, and immobilization each lowered plasma leptin and insulin levels and were associated with decrements in CRF2 receptor mRNA levels in the VMH. Peripheral injection of leptin increased VMH CRF2 receptor mRNA, as can induce reductions of food intake and body weight, indicating that circulating leptin is involved in the regulation of VMH CRF2 receptor mRNA expression. Therefore, it is also plausible that VMH CRF2 receptor transduces the anorexogenic effects of leptin as well as those of urocortins. The systemic administration of Ucn II decreases mean arterial pressure (arterial vascular tone) and causes tachycardia via vascular CRF2 receptor in rats, similar to the effects of Ucn I. Thus, CRF2 receptor seems to mediate cardioprotective effects of urocortins.     

The role of lymphoid cells in antibody-induced suppression of the fourth component of guinea pig complement

Many laboratories have demonstrated that immunoglobulin production by B cells is controlled by networks of interacting lymphocytes and their products. Our laboratory has demonstrated that complement components produced by macrophages are also regulated by networks of interacting cells and humoral factors. Treatment of mice in vivo or guinea pig cells in vitro with anticomponent antibody specifically inhibits synthesis and secretion of the component by macrophages. We have further characterized the cellular basis for in vitro suppression of the fourth component of guinea pig complement. C4 suppression has been accomplished with dispersed spleen cells as well as intact splenic fragments. This facilitated examination of the cells responsible for long-term C4 suppression. The data suggested that C4 suppression required either cell contact or sufficient concentrations of soluble factors. Long-term suppression of C4 depends upon a lymphoid cell contained in the spleen and in lymph nodes but absent or in insufficient concentration in the peritoneum. The lymphocyte that actively maintains suppression was negative for the guinea pig T-cell marker detected by the monoclonal antibody mc8BE6. Therefore, the critical cell is either another T-cell subset or non-T lymphocyte. These data demonstrate that a network of interacting cells analogous to that proposed to regulate antibody synthesis is also involved in regulating some nonlymphoid cell products.     

Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification.

Primary duck hepatocytes were infected with a mutant duck hepatitis B virus defective in envelope protein but competent for viral DNA synthesis. Cells infected by this mutant accumulated higher levels of viral covalently closed, circular DNA (cccDNA) than those infected by wild-type virus. The accumulation of high levels of cccDNA was due to a failure of the mutant-infected cells to suppress de novo cccDNA synthesis compared with suppression by cells infected by the wild type. The envelope-defective virus failed to establish a persistent infection in vitro, possibly because of a virus-mediated cell death. Therefore, one or both viral envelope proteins are required for regulation of cccDNA synthesis and for maintenance of persistent infection in vitro.     

IL-12 and neutralization of endogenous IL-10 revert the in vitro antigen-specific cellular immunosuppression of paracoccidioidomycosis patients

Treatment of patients with paracoccidioidomycosis is still a challenge. Patients present defective lymphoproliferation and IFN-gamma responses to the main Paracoccidioides brasiliensis antigen (gp43), which correlates with disease severity. Here, we demonstrated that the patients show also a defective synthesis of interleukin (IL)-12. Therefore, we attempted to revert this immune disfunction by adding IL-12 and neutralizing anti-IL-10 antibody to gp-43-stimulated peripheral blood mononuclear cell cultures. Both treatments increased IFN-gamma secretion to levels observed with healthy sensitized individuals, but affected proliferation only modestly. When combined, the treatments further increased IFN-gamma synthesis and cell proliferation. The addition of suboptimal concentrations of IL-2 also further increased the IL-12-mediated secretion of IFN-gamma. Interestingly, the immune modulation was mostly antigen-specific, since the responses to Candida albicans' antigen were not affected. These results suggest that appropriate immune intervention with cytokines and/or anti-cytokines may help in the treatment of PCM.     

Temperature is a powerful promoter of interleukin 2 transcription.

Elevated temperature has profound effects on the immune system, particularly by increasing T-cell proliferation rates, interleukin 1 (IL-1)-driven secretion of IL-2, and primary antibody responses to T-dependent antigens. Therefore, this study shows, in detail, the effects of incubation temperature (29 degrees C to 41 degrees C) on proliferation, IL-2 secretion, and IL-2 mRNA expression in both a murine thymoma cell line (EL4-6.1) and in nontransformed murine splenocytes. Temperature was found to be a positive regulator of IL-2 secretion whether or not IL-1 was part of the activation signal. Parallel effects were observed at the level of IL-2 gene expression. Messenger RNA was quantitated with a novel system, using solution hybridization followed by detection of RNA-DNA complexes by enzyme immunoassay. The time to onset of IL-2 mRNA expression was inversely related to temperature, and mRNA levels increased 20- to 50-fold with increases in average incubation temperature from 29 degrees C to 39 degrees C. This effect was observed whether cells were incubated at constant temperature or exposed intermittently to elevated temperature. Over the same intervals of time and temperature, mRNA levels for tau-actin and beta-tubulin remained relatively constant. Taken together, these findings suggest that temperature-mediated augmentation of IL-2 secretion does not require the presence of IL-1, and that the effect occurs at a pretranslational level.     

Molecular regulation of MHC class III (C4 and factor B) gene expression in mouse peritoneal macrophages

To study the molecular regulation of C4 and factor B synthesis in mouse peritoneal macrophages, mouse C4 cDNA clones isolated from an H-2d haplotype liver cDNA library, and a previously described mouse factor B cDNA clone, pBmB2 (9), were used to assess quantitative and qualitative differences in C4 and factor B mRNA in resident and elicited cells. The C4 clones that were isolated, pBmS2 (1 Kb) and pBmS10 (0.9 Kb), overlap and together span a 1.5 Kb coding region of mouse pro-C4, extending from the alpha-chain through the gamma-chain; four nucleotide substitutions are evident in comparing 316 bp of the sequence of clone pBmS10 to that of a previously described mouse C4 clone, pMLC4/w7-2 (23). By using these probes, Northern blot analysis of total cellular RNA revealed similar C4 mRNA levels in resident peritoneal macrophages from high-C4 (B10.A) and low-C4 (C3HeB) strains. Pulse and pulse-chase studies of C4 and factor B synthesis were performed on resident, starch-elicited, and thioglycollate-elicited peritoneal macrophages at two culture time periods, 0 to 9 and 24 to 33 hr, and total cellular RNA was isolated from each population at 4.5 and 28.5 hr of culture for Northern blot analysis of C4 and factor B mRNA content. The data demonstrate that as previously reported, C4 production decreases in elicited compared with resident macrophages and decreases with time in culture; however, factor B synthesis does not differ among resident and elicited cells and it increases with time in culture. The variations in C4 and factor B production by mouse peritoneal macrophages are not associated with alterations in C4 and factor B protein processing, catabolism, or secretion; rather, they are a function of differences in net amounts of C4 and factor B mRNA. These data provide direct evidence that the regulation of expression of these class III MHC genes in mouse peritoneal macrophages is a pretranslational event.