Secretion of IL-1: role of protein kinase C.

In an attempt to define the mechanism by which endotoxin induces its biologic activity, LPS was incorporated into phospholipid vesicles (liposomes) and compared with free LPS for ability to stimulate human monocytes. Activation of human monocytes by free LPS caused the translocation of protein kinase C (PKC) from the cytosol to the plasma membranes, the production of both IL-1, alpha and beta, and IL-1 secretion. Activation by LPS presented in multilamellar vesicles (MLV)-LPS caused IL-1 production but not IL-1 secretion. Moreover, MLV-LPS did not induce PKC translocation. MLV themselves did not inhibit monocyte stimulation by LPS, since LPS presented at the surface of lyophilized liposomes behaved like free LPS in cell activation. In contrast, MLV-LPS primed monocytes for subsequent LPS stimulation. When monocytes were activated by LPS in the presence of PKC inhibitors, no plasma membrane-associated PKC or IL-1 secretion was detected, whereas IL-1 production was observed. PKC inhibitors did not affect IL-1 alpha and IL-1 beta production, showing that PKC is not involved in the production of either IL-1. It can be concluded that IL-1 production and secretion are induced independently, and that IL-1 secretion involves PKC.     

IL-2 induction of IL-1 beta mRNA expression in monocytes. Regulation by agents that block second messenger pathways

We have previously shown that in mixed cultures of PBL incubation with human rIL-2 induces the rapid expression of IL-1 alpha and IL-1 beta mRNA. Because studies have demonstrated that IL-2R can be expressed on the surface of human peripheral blood monocytes, we chose to investigate whether IL-1 beta mRNA could be directly induced in purified human monocytes by treatment with Il-2 and, if so, to analyze the second messenger pathways by which it may be controlled. Human monocytes do not spontaneously express IL-1 beta mRNA, but can express the gene as soon as 1 h after treatment with IL-2. The level of IL-1 beta mRNA induced by IL-2 at 5 h in human monocytes was about one-fourth that induced by LPS. LPS induction of IL-1 beta mRNA in human monocytes can be blocked by either an inhibitor of protein kinase C (PKc) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine or an inhibitor of calcium/calmodulin (CaM) kinase N-(6-aminohexyl) 5-chloro-1-naphthalenesulfonamide, suggesting that both PKc and CaM kinase are involved in transducing signals initiated by LPS. In contrast, IL-2 induction of IL-1 beta mRNA expression is blocked only by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, suggesting that PKc, and not CaM kinase, is activated by IL-2. These data suggest that overlapping but distinct second messenger pathways are involved in the transduction of signals initiated by IL-2 and LPS.     

Induction of human monocyte IL-1 mRNA and secretion during anti-CD3 mitogenesis requires two distinct T cell-derived signals.

The T cell signals that regulate the induction of human monocyte IL-1 during primary immune activation were investigated by using anti-CD3 mitogenesis. The induction of monocyte IL-1 alpha and beta mRNA during anti-CD3 mitogenesis was rapid (less than or equal to 1 h) and required the presence of both T cells and anti-CD3. The addition of T cells plus a nonmitogenic anti-CD5 antibody failed to induce IL-1 alpha or beta mRNA, indicating that IL-1 mRNA induction by anti-CD3 required T cell activation. Experiments using double chamber culture wells revealed that the major initial phase of IL-1 alpha and beta mRNA induction (1 to 12 h) required direct cell contact between monocytes and T cells. A subsequent minor late phase (greater than or equal to 12 h) of IL-1 mRNA was induced independently of cell contact in monocytes that received only soluble factors generated during anti-CD3 mitogenesis and was temporally associated with the appearance in culture supernatants of the late phase IL-1-inducing cytokines, IL-2, IFN-gamma, and TNF-alpha. Metabolic inactivation of T cells using paraformaldehyde demonstrated that the ability of T cells to induce IL-1 mRNA via cell contact was acquired only after activation of T cells via solid phase anti-CD3. Furthermore, pretreatment of T cells with the protein synthesis inhibitor emetine had no effect on T cell-mediated induction of monocyte IL-1 mRNA or cell-associated IL-1 alpha and beta, indicating that the expression of the IL-1 inductive signal did not require protein synthesis. Despite their ability to induce monocyte IL-1 alpha and beta mRNA, activated T cells treated with paraformaldehyde or emetine were no longer able to induce monocytes to secrete IL-1 beta into culture supernatants. However, supernatants from purified T cells that were activated with solid-phase anti-CD3 restored the ability of paraformaldehyde or emetine-treated T cells to induce IL-1 secretion. These studies provide evidence that supports a two-signal model of monocyte IL-1 production during primary immune activation. The first signal leads to the induction of monocyte IL-1 mRNA and is mediated by direct contact with activated T cells, and the second signal is provided by soluble T cell factors and results in IL-1 secretion.     

Down-regulation of IL-1 beta biosynthesis by inducers of the heat-shock response

The effect of heat on IL-1 beta biosynthesis was investigated in both THP-1 cells, a myelomonocytic cell line which can be induced to make IL-1 alpha and beta, and human peripheral blood adherent monocytes (PBMC). Induction of THP-1 cells with LPS at 39 to 41 degrees C for 2 to 4 h resulted in the expected increased synthesis of the heat-shock proteins hsp 70 and hsp 90 but decreased synthesis of the IL-1 beta precursor protein, p35 (and its mRNA), compared with control cells at 37 degrees C. This appeared to be a direct effect on p35 synthesis rather than a block in LPS induction because heat also acted on preinduced cells. PBMC similarly incubated for 4 h with LPS required a temperature of 41 to 42 degrees C to induce hsp and show a decrease in p35 synthesis. Chemical inducers of the heat-shock response (heavy metals, sulphydryl reagents) were also effective inhibitors of IL-1 beta biosynthesis. A correlation was seen between the extent of IL-1 beta reduction and the level of hsp induction by chemical inducers in both THP-1 cells and PBMC which suggests that the two responses are linked. In addition, a gold salt currently used for therapy of chronic inflammation, auranofin, induced hsp and inhibited IL-1 beta biosynthesis, whereas a second salt, sodium aurothiomalate, did neither. These results support the hypothesis that elevated temperature is one of the physiologic signals for down-regulation of IL-1 beta biosynthesis through a mechanism related to the induction of hsp.     

Activated human Langerhans cells express mRNA for IL-1 alpha and IL-1 beta and produce these cytokines but do not secrete them.

Human Langerhans cells (LC) were isolated from epidermal cell preparations by panning with mouse anti-CD1 monoclonal antibody. RNA was prepared and probed for the presence of mRNAs for various cytokines using radiolabeled cDNAs. After stimulation with phorbol myristate acetate LC express RNA for interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta) and produce proteins but do not secrete them at detectable levels. LC-associated IL-1, particularly IL-1 alpha, may play a role in antigen presentation. PMA did not induce IL-6 expression in LC. The addition of lipopolysaccharide, a muramyl dipeptide analog, ionomycin, IL-1 alpha, tumor necrosis factor-alpha, insulin-like growth factor-1 or IL-6 did not induce IL-1 mRNA in LC. UVB augmented IL-1 beta mRNA expression. Glucocorticoids did not detectably affect IL-1 alpha or IL-1 beta mRNA levels following PMA induction, however, staurosporin inhibited IL-1 beta mRNA synthesis. Thus the inducers and regulators of IL-1 formation in human LC and monocytes are not identical.     

Interleukin-1 and tumour necrosis factor production by human monocytoid cells: study on a single cell level.

Human IL-1 beta and TNF alpha production by normal and transformed monocytoid cells was studied using biological assays, cytokine specific ELISA and by immunocytochemical methods on a single cell level. Quiescent human blood monocytes and cultured in vitro transformed human monocytoid cell lines U-937, THP-1 and HL-60 did not contain IL-1 beta and TNF alpha in their cytoplasm. IL-1 beta synthesis and secretion was induced by LPS stimulation in nearly 90% monocytes, 15-20% U-937, 3-5% THP-1 and in no HL-60 cells. Normal human blood monocytes had a more rapid kinetics of IL-1 beta synthesis. IL-1 beta positive cells stained with antibodies to human IL-1 beta appeared at 1-2 hours after LPS application, while in monocytic cell lines only after 4-6 hours. Using immunoperoxidase staining of U-937 cells pulse labelled with 3H-thymidine, it was shown that proliferating cells did not synthetize IL-1 beta. Instead of IL-1 beta, TNF alpha could be induced by LPS in U-937 cells only after preliminary differentiation with PMA. Recombinant IL-1 beta induced a very low level of TNF alpha production in PMA-treated cells. Similarly recombinant TNF alpha alone induced IL-1 beta synthesis only in a few U-937 cells.     

Regulation of murine mononuclear phagocyte inflammatory products by macrophage colony-stimulating factor. Lack of IL-1 and prostaglandin E2 production and generation of a specific IL-1 inhibitor

The influence of macrophage (M)-CSF on the production of inflammatory mediators has been examined in murine peritoneal macrophages. Cultures of macrophages treated with up to 30,000 U/ml of human rM-CSF or with 10,000 U/ml of L929-derived M-CSF did not reveal either PGE2, IL-1, or IL-6 secretion. In contrast, LPS, which served as a positive control, stimulated production of significant levels of PGE2, IL-1, and IL-6. Furthermore, Northern blot analysis of macrophage RNA revealed a strong induction of IL-1 alpha and IL-6 mRNA by LPS but not by M-CSF. Conditioned medium from macrophage cultures treated with purified L929 or human rM-CSF in combination with LPS exhibited a significant reduction of IL-1 bioactivity as compared with an LPS challenge alone. To investigate the mechanism involved in this M-CSF-dependent reduction of IL-1 bioactivity, we measured IL-1 alpha gene expression. The addition of M-CSF to LPS-treated macrophages did not affect IL-1 alpha mRNA levels suggesting that M-CSF may regulate production of an IL-1 inhibitor. This hypothesis was shown to be valid because removal of IL-1 alpha from conditioned medium of LPS plus M-CSF-treated cells allowed the detection of a nondialyzable factor that blocked IL-1-dependent thymocyte proliferation. The inhibitor appeared to be specific because it did not inhibit IL-2 and TNF bioactivities. Furthermore, this IL-1 inhibitor, which binds to cells and not to IL-1, competed with the binding of radioactive IL-1 alpha or beta to EL-4.6.1 cells. The results demonstrate that M-CSF alone does not induce proinflammatory mediators and PGE2 as was previously published. The data also suggest that M-CSF may play a role in the down-regulation of inflammatory responses.     

IL-1 gene expression in lymphoid tissues

We examined the expression of IL-1 mRNA in vivo by in situ hybridization. RNA probes for murine IL-1 alpha and IL-1 beta were used to detect IL-1 mRNA in frozen sections of spleen, lymph node, and thymus of mice injected with Salmonella typhi LPS or SRBC. No IL-1 was detected in lymphoid tissues from un-injected mice. This lack of expression correlated with the absence of IL-1 biologic activity. However, after LPS injection, IL-1 alpha and beta mRNA expression was found in macrophages of the red pulp and marginal zone of the spleen. The periarteriolar lymphoid sheath contained cells that only expressed IL-1 beta mRNA. These cells were not lymphocytes and did not stain with the macrophage marker F4/80. A similar cellular response was found after SRBC injection. Scattered macrophages in lymph nodes and thymus were positive, but only after LPS or SRBC injection. The spleens of mice injected with LPS had megakaryocytes containing IL-1 mRNA.     

alpha1-Antitrypsin regulates CD14 expression and soluble CD14 levels in human monocytes in vitro

The recognition of bacterial lipopolysaccharide (LPS) is principally mediated by either membrane-bound or soluble form of the glycoprotein CD14 and CD14-associated signal transducer, toll-like receptor 4 (TLR4). Recent findings indicate that the serine protease inhibitor, alpha1-antitrypsin (AAT), may not only afford protection against proteolytic injury, but may also neutralize microbial activities and affect regulation of innate immunity. We postulated that AAT affects monocyte responses to LPS by regulating CD14 expression and soluble CD14 release. Here we show that a short-term (up to 2h) monocyte exposure to AAT alone or in combination with LPS leads to a remarkable induction of CD14 levels. In parallel, a short-term (2h) cell exposure to AAT/LPS significantly enhances LPS-induced NF kappaB (p50 and p65) activation in conjunction with increased TNFalpha, IL-1 beta and IL-8 release. In contrast, longer term incubation (18 h) of monocytes with combined AAT/LPS results in a significant reduction in expression of both CD14 and TLR4, inhibition of LPS-induced TNFalpha, IL-1 beta and IL-8 mRNA and protein expression. These findings provide evidence that AAT is an important regulator of CD14 expression and release in monocytes and suggest that AAT may be involved in LPS neutralization and prevention of over-activation of monocytes in vivo.