Recombinant human interleukin 1-stimulated Na+/H+ exchange is not required for differentiation in pre-B lymphocyte cell line, 70Z/3

Interleukin-1 a polypeptide hormone produced by activated macrophages is a mixture of at least two proteins, interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta). We have previously shown that macrophage-derived interleukin-1 induced new kappa light chain synthesis for surface IgM expression in a murine pre-B like cell line 70Z/3, a finding associated with an early amiloride-sensitive rise in the total intracellular sodium concentration. Because IL-1 alpha and IL-1 beta are structurally quite different, in this study their effect on 70Z/3 was examined separately. The results show that both human rIL-1 alpha and rIL-1 beta induce the differentiation of 70Z/3, but a higher concentration of rIL-1 beta compared to rIL-1 alpha is needed for a maximal response. At saturating concentrations, both rIL-1 alpha and rIL-1 beta induce a simultaneous rise in intracellular pH and sodium concentration. Because rIL-1 mediated intracellular alkalinization and sodium rise are amiloride sensitive, they likely occur through stimulation of the Na+/H+ exchanger across the cell membrane. Inhibition of the Na+/H+ antiport with an amiloride analog did not have an effect on rIL-1 induced surface IgM expression or the rIL-1-mediated increase in kappa light chain specific mRNA level. Therefore, these results indicate that an increase in pHi or [Na]i is not required for IL-1 induced 70Z/3 differentiation.     

Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo.

The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines. The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood. All CXC (IFN-gamma-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1alpha, MIP-1beta, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo. While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-gamma selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1alpha, and MIP-1beta mRNA and/or protein. Like the response to IFN-gamma, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent. Interestingly, only the IFN-gamma-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent. Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein. Hepatic expression of MIP-1alpha, MIP-1beta, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced. These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.     

The effect of LPS on expression of the early "competence" genes JE and KC in murine peritoneal macrophages

The expression of early "competence" genes has been examined in murine peritoneal macrophages treated with bacterial lipopolysaccharide (LPS). This set of genes (e.g., c-myc, c-fos, r-fos, JE, and KC) were first described in BALB/c 3T3 cells treated with platelet-derived growth factor. We have previously reported that LPS induces the rapid and transient expression of both c-myc and c-fos in macrophages. In the present report, we present evidence demonstrating that the mRNA for JE and KC are also induced in macrophages after treatment of LPS. The r-fos gene was not detectably induced by LPS under the experimental conditions used in this study. The induction of JE and KC were dependent upon the dose of LPS and exhibited different time courses. mRNA for both KC and JE was induced within 30 min from the initiation of treatment. Although mRNA for JE continued to accumulate for up to 24 hr, mRNA for KC was optimally seen after 60 min and had disappeared by 4 hr. c-fos, JE, and KC mRNA were all inducible by a variety of structurally diverse but functionally similar agents (e.g., heat killed Listeria monocytogenes, maleyl-bovine serum albumin, and fucoidan). Interferon-gamma, a potent but functionally distinct stimulus of macrophage activation, did not effect the expression of JE or KC mRNA. The expression of mRNA for c-fos could be readily induced by treatment of macrophages with phorbol myristate acetate (PMA) alone and that for JE by PMA plus the inophore A23187; mRNA for KC was largely unaffected by these agents. These results suggest that expression of the c-fos and JE genes are regulated by products of polyphosphoinositide hydrolysis. The difference between c-fos or JE and KC raises the possibility that LPS may stimulate at least two independent routes of early gene expression. LPS does not promote macrophage proliferative activity alone, and in fact inhibits the proliferative response to the macrophage growth factor colony-stimulating factor 1. Taken together these findings suggest that the products of these genes may function in the acquisition of competence for highly differentiated functions in addition to that for cell division.     

Anaphylatoxin C5a actions in rat liver: synergistic enhancement by C5a of lipopolysaccharide-dependent alpha(2)-macroglobulin gene expression in hepatocytes via IL-6 release from Kupffer cells

The effects of the anaphylatoxins C5a and C3a on the liver are only poorly characterized in contrast to their well known systemic actions. Recently, it has been demonstrated that the anaphylatoxin C5a enhanced glucose output from hepatocytes (HC) indirectly via prostanoid release from Kupffer cells (KC). In the present study, it is shown that recombinant rat C5a (rrC5a), together with LPS, activated the gene of the acute phase protein alpha(2)-macroglobulin (alpha(2)MG) in HC also indirectly via IL-6 release from KC. RrC5a alone increased neither IL-6 mRNA in nor IL-6 release from KC, whereas LPS alone did so. However, rrC5a synergistically enhanced the LPS-dependent increase in IL-6 mRNA and IL-6 release. Only rIL-6, but not TNF-alpha or IL-1beta, enhanced alpha(2)MG mRNA in HC. In line with the actions of rrC5a and LPS on KC, conditioned medium of KC stimulated only with rrC5a did not increase alpha(2)MG mRNA in HC. However, medium of KC stimulated with rrC5a plus LPS induced alpha(2)MG mRNA expression in HC more strongly than medium from cells stimulated only with LPS; thus, C5a acted synergistically with LPS. The stimulatory effects of KC-conditioned medium could partially be inhibited by a neutralizing anti-IL-6 Ab, indicating that KC-derived IL-6 was a major mediator in C5a- plus LPS-elicited alpha(2)MG gene expression. These results suggest that C5a, besides enhancing glucose output via prostanoids, is involved in the initiation of the acute phase response in HC via proinflammatory cytokines from KC. This provides evidence for another important function of C5a in the regulation of hepatocellular defense reactions.     

Molecular cloning of gene sequences regulated by platelet-derived growth factor

We have screened a cDNA library for gene sequences that are regulated by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells. Of 8000 clones screened, less than 14 independent PDGF-inducible sequences were found. Two of these (KC and JE) were studied in detail. By hybrid-selection and translation the KC and JE mRNAs encode 10,000 and 19,000 dalton polypeptides, respectively. In the absence of PDGF, the JE and KC sequences correspond to low abundance mRNAs. One hour after addition of PDGF their abundance level can be increased 10- to 20-fold. Within 4 hr, a 60-fold induction of JE can be attained. Nanogram per ml quantities of pure PDGF regulate these sequences whereas μg/ml quantities of chemically unrelated mitogens (EGF, insulin, or platelet-poor plasma) have either a weak or an undetectable effect. Inhibitors of protein synthesis block the progression of quiescent 3T3 cells through G1 into S phase; however these drugs do not block the induction of KC and JE by PDGF. This result indicates that these sequences correspond to “early genes” which are not induced as a consequence of cell growth, but rather are directly regulated by PDGF.     

The interleukin-1 receptor antagonist influences interleukin-1 effects in rat and mouse

In this work we have focused on the ability of interleukin-1 to induce an acute phase protein response and a degranulation of polymorphonuclear leukocytes in vivo. The capacity of the interleukin-1 receptor antagonist to influence these events was also investigated. It was shown that interleukin-1 induced an acute phase protein response in rats and mice. In rats alpha(2)-macroglubolin levels were increased in plasma after an interleukin-1 injection whereas alpha(1)-inhibitor-3 decreased in plasma. In the mice plasma amyloid P was increased. The interleukin-1 receptor antagonist blocked the increase of alpha(2)-macroglobulin and plasma amyloid P in a dose dependent way while the effect on the alpha(1)-inhibitor-3 decrease was less pronounced. Interleukin-1 led to polymorphonuclear leukocyte degranulation in vivo as measured by increased cathepsin G plasma levels. The interleukin-1 receptor antagonist could influence the early phase of this degranulation.     

Tumor necrosis factor-alpha enhances inhibitory effects of interleukin-1 beta on Leydig cell steroidogenesis

Human recombinant tumor necrosis factor-alpha (rTNF alpha) alone (up to 1000 units/ml) did not alter either basal or human chorionic gonadotropin (hCG)-induced testosterone formation in primary culture of rat Leydig cells. However, concomitant addition of rTNF alpha with human recombinant interleukin-1 beta (rIL-1 beta) enhanced the inhibitory effects of rIL-1 beta. The rIL-1 beta dose response curve was shifted to the left (IC50 changed from 1 ng/ml to 0.3 ng/ml). Even though rTNF alpha had no effect on testosterone formation, hCG-stimulated cyclic AMP formation was inhibited by rTNF alpha in a dose dependent manner. In the presence of both rTNF alpha and rIL-1 beta, hCG-induced cyclic AMP formation and binding of [125I]-hCG to Leydig cells were further inhibited. Testicular macrophages represent about 20% of the interstitial cells. TNF alpha and IL-1 may be produced locally by interstitial macrophages and have paracrine effects on Leydig cell function.     

Species differences in human and rat islet sensitivity to human cytokines. Monoclonal anti-interleukin-1 (IL-1) influences on direct and indirect IL-1-mediated islet effects

Species differences in sensitivity to human recombinant cytokines were observed when human or rat islets were co-cultured with human recombinant cytokines for 6 days. Suppression of both human and rat islet insulin secretion resulted from co-culture with recombinant interleukin-1 alpha (rIL-1 alpha) or interleukin-1 beta (rIL-1 beta); however, direct rIL-1 alpha and rIL-1 beta cytotoxicity was seen with rat islets but not with human islets. Human islet insulin secretion was also suppressed during co-culture with recombinant tumor necrosis factor (rTNF) or interferon (rIFN), but not with lymphotoxin (rLT) or rIL-6; rat islet insulin secretion was not suppressed by any of these cytokines. No direct cytotoxic effects resulted from co-culture of human islets with rLT, rTNF, rIFN, or rIL-6; rLT was slightly cytotoxic for rat islets. Human islet cytotoxic synergy occurred between rLT and rIL-1 alpha, rIL-1 beta, or rIFN; synergy in suppression of human islet insulin secretion occurred between rLT and rIL-1 beta, and between rIFN and rTNF. Pretreatment of rIL-1 with monoclonal antibody (mAb) specific for non-crossreactive epitopes on rIL-1 alpha (H43 and H12) or rIL-1 beta (H34 and H21) prevented islet cytotoxic synergy between rIL-1 alpha or rIL-1 beta, respectively, and rLT. Although all four mAb's neutralize the thymocyte and fibroblast stimulatory activities of rIL-1 alpha or rIL-1 beta, mAb H21 does not neutralize rIL-1 beta activity against rat islets. Implications for cytokine-mediated islet cytotoxicity and suppression of insulin secretion are discussed.     

Potent inhibition of gastric acid secretion by intravenous interleukin-1 beta and -1 alpha in rats.

The influence of human and rat recombinant interleukin-1 (hIL-1 beta and -1 alpha and rIL-1 beta) on acid secretion was investigated in conscious pylorus-ligated rats. Intravenous injection of either hIL-1 beta, hIL-1 alpha or rIL-1 beta dose dependently inhibited gastric acid output with an ED50 of 0.05 microgram, 0.5 microgram and 2.2 micrograms, respectively. The antisecretory action of IL-1 beta was associated with an increase in circulating levels of gastrin. hIL-1 beta-induced inhibition of acid secretion was dose dependently reversed by peripheral injection of the IL-1 receptor antagonist, IL-RA, with a dose ratio of 1:10(3) for complete reversal. The inhibitory effect of hIL-1 beta was blocked by indomethacin and was not modified by IV injections of the CRF receptor antagonist, alpha-helical CRF(9-41), or the monoclonal somatostatin antibody CURE.S6, or by systemic capsaicin pretreatment. These results show that systemic hIL-1 beta-induced inhibition of gastric acid secretion is mediated through IL-1 receptors and prostaglandin pathways, and does not involves CRF receptors, afferent fibers, or changes in circulating gastrin or somatostatin levels.     

The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes

During the acute phase response, synthesis of C-reactive protein and serum amyloid A is increased. To investigate whether the enhanced synthesis of these proteins are due to stimulatory effect of inflammatory mediators such as interleukin-1 (IL-1) and interleukin-6 (IL-6) produced by macrophages and monocytes, primary cultures of adult human hepatocytes were exposed to recombinant (r)IL-1, rIL-6 or rIL-1 and monospecific anti rIL-6 antibodies in the presence of 1 microM dexamethasone. The findings indicate that rIL-1 and rIL-6 both stimulate the liver synthesis of C-reactive protein and serum amyloid A, however monospecific anti rIL-6 antibodies reduce the stimulatory effect of rIL-1 on the synthesis of these proteins. These findings suggest that IL-6 plays a key role in the stimulation of synthesis of serum amyloid A and C-reactive protein by the human liver cells.