IL-1alpha mRNA Levels Research Articles

Interleukin-1 alpha gene expression and localization of interleukin-1 alpha protein during tumor promotion.

Treatment of the dorsal epidermis of SENCAR mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and dose-dependent stimulation of interleukin-1 alpha (IL-1 alpha) gene expression. Levels of IL-1 alpha mRNA were elevated as early as 15 min and peaked at 3-4 h after a single application of TPA (2 micrograms or 10 micrograms). IL-1 alpha gene expression increased in epidermal tissue isolated from SENCAR mice at 1, 3, 6, 10, 16, and 22 wk after a single treatment with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and subsequent twice-weekly application of 2 micrograms TPA. IL-1 alpha-immunoreactive protein was specifically localized within suprabasal keratinocytes in cutaneous tissue isolated from mice treated with DMBA-TPA for 1-22 wk and in nonproliferating cells located within papilloma tissue isolated from SENCAR mice at 22 wk after initiation and promotion. Basal cells within hyperplastic epidermis did not produce IL-1 alpha-immunoreactive protein. DMBA treatment alone did not induce IL-1 alpha gene expression. Injection of IL-1 alpha-specific antibodies (50 micrograms) into SENCAR mice via the tail vein 2 h before treatment with TPA (2 micrograms or 10 micrograms) significantly (P < 0.05) inhibited the skin thickening usually observed 24 h after treatment with TPA. Autoradiography studies showed that injection of anti-IL-1 alpha antibodies inhibited incorporation of [methyl-3H]thymidine by keratinocytes within the epidermis and by cells within hair follicles. It also inhibited neutrophil infiltration into the dermis, which usually results from topical application of TPA. These data suggest that IL-1 alpha is a pivotal cytokine produced by specific subpopulations of epidermal keratinocytes and that IL-1 alpha primarily regulates the epidermal proliferative response of a distinctly separate population of keratinocytes after topical exposure of murine epidermis to TPA and secondarily modulates neutrophil migration into the dermis. Consequently, manipulation of IL-1 alpha may be a way to attenuate or abrogate the cutaneous response to TPA by altering keratinocyte proliferation, the resultant hyperplasia, and a portion of the inflammatory response characterized by dermal infiltration of neutrophils.

Insulin-Dependent Diabetes in the Nod Mouse Model II. β Cell Destruction in Autoimmune Diabetes is a TH2and not A TH1Mediated Event

Type I, insulin-dependent diabetes (IDD) in both man and animals results from a specific autoimmune destruction of the pancreatic beta cells involving both humoral and cellular immune mechanisms. The pathognomonic histologic lesion, termed insulitis, is an inflammatory and immune cell infiltrate of the pancreatic islet cells. While recent histological and flow cytometric analyses have identified the cell composition of the infiltrate, the presence of a cell population may not reflect the functional reactivities important for beta cell destruction. In the present study, we have investigated the possible functional reactivities of islet-infiltrating mononuclear cell populations by measuring increased cytokine mRNA usage. Results indicate that 1) cytokine mRNA profiles exhibited by islet-infiltrating cells of female and male NOD mice were quite similar with the exception of IL-6 expression and the marked differences in the levels of IL-2 receptor and IL-1 alpha mRNA, 2) CD4+ T lymphocytes expressed IL-4, presumably IL-5, and occasionally IL-10 mRNA but no detectable IL-2 mRNA, 3) CD8+ T lymphocytes exhibited TNF-beta, perforin and high levels of IFN-gamma, and 4) IL-7 was expressed in the islet at very high levels. These findings, together with our earlier flow cytometric analyses of the islet-infiltrating cells, have permitted construction of a detailed model for the natural history of autoimmune diabetes. Interestingly, this model, based on a TH2- and not a TH1-mediated scheme, questions the more popular concepts currently thought to form the bases of the autoimmune reactions underlying IDD.

Contrasting mechanisms for suppression of macrophage cytokine release by transforming growth factor-beta and interleukin-10

Transforming growth factor (TGF)-beta and interleukin (IL)-10 inhibited lipopolysaccharide (LPS)-induced macrophage production of the inflammatory cytokines tumor necrosis factor-alpha (TNF), IL-1 alpha, and IL-1 beta by contrasting post-transcriptional mechanisms. TGF-beta acted slowly and late, as it required 12-16 h to exert a suppressive effect, and inhibited TNF production even when added 6 h after LPS. TGF-beta affected neither the level of TNF mRNA, the release of preformed TNF nor the degradation of TNF. Thus, TGF-beta appeared to inhibit translation of TNF mRNA. IL-10 not only suppressed TNF release to a 25-fold greater extent than TGF-beta, but also inhibited release of IL-1. In contrast to TGF-beta, IL-10 acted on an early step in cytokine production, its effect being maximal 3 h after addition of LPS. Unlike TGF-beta, IL-10 markedly suppressed TNF, IL-1 alpha, and IL-1 beta mRNA levels. However, this was accomplished without suppressing transcription of the corresponding genes. Moreover, cycloheximide antagonized the IL-10-dependent reduction in cytokine mRNA levels. Thus, IL-10 may induce a ribonuclease active on cytokine transcripts or may induce a protein that enhances the susceptibility of TNF, IL-1 alpha, and IL-1 beta mRNAs to ribonucleolytic action. We conclude that IL-10 and TGF-beta induce different phenotypes of macrophage deactivation, and deactivate macrophages by different mechanisms: IL-10 promotes degradation of cytokine mRNA, while TGF-beta primarily suppresses translation.

Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery.

Experimental autoimmune encephalomyelitis (EAE) serves as an important animal model for understanding the events that lead to immune-mediated inflammation and tissue destruction within the central nervous system. We have utilized a murine adoptive transfer model of EAE and semiquantitative reverse transcriptase-polymerase chain reaction analysis to examine cytokine mRNA expression within the central nervous system in relation to the onset and resolution of paralysis associated with EAE. Spinal cord samples, obtained from mice as they progressed through discrete clinical stages of EAE, were examined for the expression of six cytokine genes (IL-1 alpha, IL-2, IL-4, IL-6, IL-10, and IFN-gamma). Distinct patterns of cytokine gene expression were observed during the acute, recovery, and chronic phases of EAE. The acute phase of disease was characterized by rapid increases in the levels of mRNA for IL-2, IL-4, IL-6, IFN-gamma, and IL-1 alpha. In fact, peak expression of several cytokine mRNA (e.g., IL-2, IL-4, IL-6, and IFN-gamma) occurred before the peak in clinical severity. In contrast, IL-1 alpha mRNA levels were elevated throughout the initial disease course. IL-10 mRNA demonstrated only modest increases during the acute phase of EAE. Stabilization of the clinical symptoms was characterized by rapid declines in the mRNA levels of IL-2, IL-4, IL-6, and IFN-gamma. The decreases in these four cytokine mRNA levels occurred concomitant with a dramatic rise in IL-10 mRNA. Finally, of the six cytokine mRNA examined, only IL-1 alpha, IFN-gamma, and IL-10 mRNA remained elevated during the early chronic stage. These results suggest that local cytokine production varies significantly during the course of EAE and that increases in discrete sets of cytokines are associated with the acute response and the recovery/chronic phase of disease.

Cutaneous barrier perturbation stimulates cytokine production in the epidermis of mice.

The disruption of the cutaneous permeability barrier results in metabolic events that ultimately restore barrier function. These include increased epidermal sterol, fatty acid, and sphingolipid synthesis, as well as increased epidermal DNA synthesis. Because tumor necrosis factor (TNF) and other cytokines are known products of keratinocytes and have been shown to modulate lipid and DNA synthesis in other systems, their levels were examined in two acute models and one chronic model of barrier perturbation in hairless mice. Acute barrier disruption with acetone results in a 72% increase in epidermal TNF 2.5 h after treatment, as determined by Western blotting. Furthermore, epidermal TNF mRNA was elevated ninefold over controls 2.5 h after acetone treatment. This elevation in TNF mRNA was maximal at 1 h after acetone, and decreased to control levels by 8 h. After tape stripping, a second acute model of barrier disruption that avoids application of potentially toxic chemicals, TNF mRNA was elevated fivefold over controls at 2.5 h. Moreover, the mRNA levels for epidermal IL-1 alpha, IL-1 beta, and granulocyte macrophage-colony-stimulating factor (GM-CSF) also were elevated several-fold over controls, after either acetone treatment or tape stripping, but their kinetics differed. GM-CSF mRNA reached a maximal level at 1 h after acetone, while IL-1 alpha and IL-1 beta were maximal at 4 h after treatment. In contrast, mRNAs encoding IL-6 and IFN gamma were not detected either in control murine epidermis or in samples obtained at various times after tape stripping or acetone treatment. The relationship of the cytokine response to barrier function is further strengthened by results obtained in essential fatty acid deficient mice. In this chronic model of barrier perturbation mRNA levels for epidermal TNF, IL-1 alpha, IL-1 beta, and GM-CSF were each elevated several-fold over controls. These results suggest that epidermal cytokine production is increased after barrier disruption and may play a role in restoring the cutaneous permeability barrier.

Activation of cytokine genes in HIV-1 infected myelomonoblastic cells by phorbol ester and tumor necrosis factor.

The differential production of inflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha) was analyzed in the PLB-985 myelomonoblastic cell line, chronically infected or not by the IIIB strain of HIV-1. After treatment with phorbol ester (PMA) or TNF-alpha, a 20- to 40-fold increase in the level of IL-1 beta mRNA was observed in the HIV-infected PLB-IIIB as compared with the parental PLB-985 cells. The majority of the IL-1 beta activity detected in both cell types remained cell associated. In contrast, TNF-alpha mRNA levels were increased in both infected and uninfected cells; the t1/2 of TNF RNA was 90 min in uninfected cells and 30 min in HIV-infected cells. Interestingly, about 14-fold more TNF activity was secreted from PLB-IIIB than from similarly stimulated PLB-985 cells, indicating an enhanced translational efficiency of TNF RNA in PLB-IIIB cells. The PMA- or TNF-induced levels of IL-1 alpha mRNA did not vary significantly between the two cell types whereas IL-6 was poorly inducible in both cells. These results illustrate a differential cytokine response to HIV-1 infection in myeloid cells and demonstrate that HIV-1 infection of myelomonoblastic cells may alter both transcriptional and translational mechanisms controlling cytokine expression.

Ca2+ and calmodulin selectively regulate lipopolysaccharide-inducible cytokine mRNA expression in murine peritoneal macrophages.

The role of Ca2+ and Calmodulin in regulating LPS-induced cytokine gene expression in murine peritoneal macrophages has been investigated. Treatment of macrophages with three structurally distinct antagonists of Calmodulin (Trifluoperazine, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide, and 1,3-dihydro-1-(-((4-mentyl-4H,6H-pyrrolo(1,2-a) (4,1)-benzoxazepin-4-yl)methyl)-4-peperidinyl)-2H-benzimi dazol-2-one) resulted in a characteristic modulation of the expression of three LPS-inducible cytokine genes: IL-1 alpha mRNA levels were only modestly reduced, IL-1 beta mRNA levels were markedly suppressed and IP-10 mRNA levels were increased. The same pattern of modulation was seen when LPS-stimulated cells were also treated with two different Ca2+ antagonists (8-(diethylamono)-octyl-3,4,5-trimethoxybenzoate hydrochloride and bis-(o-amonophenoxy)-ethane-N,N,N'N'-tetraacetic acid). Although the suppression of IL-1 beta mRNA accumulation by N-(6-amonohexyl)-5-chloro-1-napthalene-sulfonamide or bis-(o-amonophenoxy)ethane-N,N,N'N'-tetraacetic acid occurred even if the antagonist was added after LPS, the potentiation of IP-10 mRNA levels required the use of the agent before or along with the LPS stimulus. Elevation of intracellular Ca2+ using ionomycin did not initaite cytokine gene expression and thus changes in Ca2+ cannot replace the LPS-initiated signal. Furthermore, removal of extracellular Ca2+ did not block the response to LPS. Calmodulin antagonists selectively increased the transcriptional activity of the IP-10 gene but decreased the stability of all three mRNA measured. Thus the mechanisms involved in Ca2+/Calmodulin control of macrophage gene expression are multifactorial and contribute to the diversity of macrophage inflammatory behavior. In concert with previous reports, the present results indicate that Ca2+, acting through Calmodulin may be a necessary but insufficient component of the signalling process that mediates intracellular response to LPS. Agents that alter intracellular Ca2+ levels without inducing cytokine gene expression may thereby indirectly regulate inflammation.

Regulation of cytokine production in the human thymus: epidermal growth factor and transforming growth factor alpha regulate mRNA levels of interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6 in human thymic epithelial cells at a post-transcriptional level.

Human thymic epithelial (TE) cells produce interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6, cytokines that are important for thymocyte proliferation. The mRNAs for these cytokines are short-lived and are inducible by multiple stimuli. Thus, the steady-state levels for IL-1 and IL-6 mRNAs are critical in establishing the final cytokine protein levels. In this study we have evaluated the effect of epidermal growth factor (EGF), a growth factor for TE cells, and its homologue transforming growth factor alpha (TGF-alpha), on primary cultures of normal human TE cells for the levels of IL-1 alpha, IL-1 beta, IL-6, and TGF-alpha mRNA. We showed that TE cells expressed EGF receptors (EGF-R) in vitro and in vivo, and that treatment of TE cells with EGF or TGF-alpha increased IL-1 and IL-6 biological activity and mRNA levels for IL-1 alpha, IL-1 beta, and IL-6. Neither EGF nor TGF-alpha increased transcription rates of IL-1 alpha, IL-1 beta, and IL-6 genes, but rather both EGF and TGF-alpha increased cytokine mRNA stability. By indirect immunofluorescence assay, TGF-alpha was localized in medullary TE cells and thymic Hassall's bodies while EGF-R was localized to TE cells throughout the thymus. Thus, TGF-alpha and EGF are critical regulatory molecules for production of TE cell-derived cytokines within the thymus and may function as key modulators of human T cell development in vivo.

Imbalanced cytokine production by macrophages from autoimmune-prone mice

Peritoneal macrophages from multiple autoimmune-prone strains of mice (MRL/lpr, MRL/+, NZB/W, BXSB, and B6/gld) show defective expression of the cytokines IL-1 and IL-6. Autoimmune mice were all used between 1 and 6 weeks of age, the earliest times being well before the onset of overt disease. Northern blot analysis reveals a parallel reduction in the levels of IL-1 alpha, IL-1 beta, and IL-6 mRNA. In contrast, the production of other proteins, including the cytokine TNF-alpha, appears to be normal. These findings imply an imbalance within the cytokine network of autoimmune macrophages. Studies on bone marrow-derived macrophage precursors, as well as macrophages from chimeric mice, suggest an intrinsic macrophage defect as opposed to conditioning by the autoimmune environment. This defect appears to be constant throughout the lifespan of autoimmune MRL/lpr mice, being equally apparent in 1-week old mice as in fully diseased 6-12-month-old mice. Aberrant regulation of IL-1 and IL-6 represents a novel defect in the function of autoimmune macrophages that is both intrinsic and substantial, and has the potential to contribute to the immune dysregulation that characterizes autoimmunity.

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.

Different Activation Signals are Required for the Expression of Interleukin‐1 α and β Genes in Human Monocytes

The production and mRNA expression of IL-1 alpha and IL-1 beta by human monocytes was examined after two different stimuli, a protein kinase C (PKC) activator phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS). LPS induced production of high levels of both IL-1 alpha and IL-1 beta protein (quantitated with type-specific ELISA assays), while after PMA stimulation only IL-1 beta protein could be detected. The IL-1 alpha and IL-1 beta mRNA levels quantitated by Northern blotting were in line with the respective protein levels and nuclear run off analysis revealed that PMA did not activate the IL-1 alpha transcription. The production of the IL-1 alpha and IL-1 beta protein as well as the mRNA expression could be inhibited with protein kinase inhibitor H7, but not with HA1004, indicating that PKC activation is essential for the activation of these genes. Thus these data indicate that PKC activation alone is sufficient for the induction of the IL-1 beta gene, but some additional signals (provided by LPS) are required for the activation of the IL-1 alpha gene.

Analysis of IL-1 and TNF-alpha gene expression in human rheumatoid synoviocytes and normal monocytes by in situ hybridization.

Human IL-1 alpha, IL-1 beta, and TNF-alpha mRNA expression was examined in peripheral blood monocytes (PBM) from normal individuals and in primary synoviocytes isolated from patients with rheumatoid arthritis by Northern blot and in situ hybridization. Cells cultured in the presence or absence of LPS were analyzed using in vitro synthesized 35S-labeled sense or antisense RNA probes to determine the relative abundance and the cell type expressing each of the mRNA for these potent inflammatory mediators. The results indicated that 72% of the LPS-stimulated PBM expressed detectable levels of IL-1 alpha mRNA, 89% IL-1 beta mRNA, and 10% TNF-alpha transcripts. Thus, the majority of activated PBM produced both IL-1 alpha and IL-1 beta. Experiments combining immunofluorescence for IL-1 beta protein with in situ hybridization for TNF-alpha mRNA demonstrated that monocytes expressing TNF-alpha mRNA also produced IL-1 beta. Primary synoviocytes from four patients with RA were also examined for the mRNA expression of each cytokine. Northern blot analyses of total RNA isolated from 0 to 72 h after LPS- or mock-stimulation showed that IL-1 beta mRNA was the most abundantly expressed, followed by TNF-alpha. In situ hybridization revealed that IL-1 beta and TNF-alpha transcripts were detected exclusively in synovial tissue macrophages. IL-1 alpha mRNA was not detected in these cultures by either method.

Interleukin-1 and Interleukin-6 in Psoriasis

We report on the levels of expression of IL-1 and IL-6 in skin from psoriasis patients. Different approaches were pursued. Initially, the levels of IL-1 beta and IL-6 were measured in suction blister fluid from lesional and uninvolved skin from psoriasis patients, using a sensitive enzyme-linked immunoabsorbent assay (ELISA) and bio-assay. Skin sections were also examined for the presence of IL-1 and IL-6 using IL-1 beta- and IL-6-specific antibodies. Finally, the expression of IL-1 and IL-6 mRNA was determined in cultured keratinocytes (KC) and fibroblasts from psoriasis skin. Suction blister fluid from lesional and uninvolved psoriasis skin and from skin of healthy individuals did not contain detectable levels (greater than 100 pg/ml) of IL-1 beta. Blister fluid from psoriasis lesions contained low but significant levels of IL-6, whereas the serum levels of IL-6 in these patients was undetectable. Using cryostat skin sections and an IL-1 beta-specific monoclonal antibody (MoAb) in an indirect immunoperoxidase technique, a diffuse staining in the entire epidermis was observed in sections of uninvolved skin from psoriasis patients. In cryostat sections of psoriasis lesions, a faint diffuse staining of the epidermis and a pronounced "dot-like" intracellular staining pattern was observed. On the other hand, the same IL-1 beta-specific MoAb showed, in a indirect immunofluorescence technique using unfixed epidermal cells, bright membrane staining in epidermal cell suspensions from psoriasis lesions. Slightly elevated levels of IL-1 beta and IL-1 alpha mRNA were observed in cultured KC from psoriasis lesions as compared to those in normal KC and in the HEp-2 cell line. Very low levels of IL-6 mRNA were expressed in KC from psoriasis lesions and healthy individuals. Fibroblasts from psoriasis lesions expressed extremely low levels of IL-1 alpha and IL-1 beta, but high levels of IL-6 mRNA. The results point to a paradoxical situation in psoriatic skin: blister fluid from psoriasis lesions contains no IL-1 beta, whereas IL-1 beta is overexpressed on the plasma membrane and in the intracellular compartment of epidermal cells. This finding may help in explaining the observed absence of IL-1 in aqueous extracts of psoriatic scales. Because cultured KC from psoriasis lesions express minimal levels of IL-6 mRNA. dermal fibroblasts, probably together with the inflammatory infiltrate, may represent a major source of IL-6 in psoriasis lesions in vivo.

The kinetics of gene expression and maturation of IL-1 alpha after induction with the surface coat of Trypanosoma brucei rhodesiense or lipopolysaccharide.

The purpose of this study was threefold: to determine if the variant surface coat glycoprotein (VSG) of Trypanosoma brucei rhodesiense induces IL-1 alpha; to study the kinetics of IL-1 alpha transcription, maturation and secretion; and to compare VSG to LPS in its ability to induce IL-1 alpha. VSG was added to cultures of the P388D1 murine macrophage cell line. RNA was dotted onto nitrocellulose and hybridized with a murine IL-1 alpha cDNA probe. Maximal production of IL-1 alpha mRNA occurred in a dose- and time-dependent manner, peaking at 25 micrograms/ml VSG, within 2 h. Induction of IL-1 alpha was not due to contaminants because 1) absorption of VSG with a mAb abrogated IL-1 alpha mRNA synthesis, 2) the addition of polymyxin B did not affect mRNA levels, and 3) cellular IL-1 alpha was detectable in VSG-treated splenocytes from endotoxin nonresponder C3H/HeJ mice. Murine splenic macrophages also had enhanced levels of IL-1 alpha mRNA after administration of VSG in vivo or during an acute infection. Antiserum generated against the synthetic peptide SGDDSKYPV (amino acids 177-185 from the murine IL-1 alpha sequence) was used to measure the levels of the 33-, 22-, and 14-kDa proteins in cell lysates and medium of VSG-stimulated P388D1 cells. The 22-kDa protein was the predominant cellular form until secretion started. Secretion of the 14-kDa form began abruptly 6 to 8 h after the addition of VSG. By 12 h, the 33-kDa precursor was the major cytoplasmic form. In comparative analyses, LPS-stimulated P388D1 cells produced more transcript, generated peak levels of 22-kDa protein 3 h earlier, and began to secrete the 14-kDa molecule 5 h earlier. The rate of IL-1 alpha accumulation in the medium was linear between 6 and 24 h after LPS treatment, but began to drop by 8 h in VSG-treated cells. Functional (comitogenic) IL-1 activity was also detected in media from VSG-treated splenic macrophages and P388D1 cells. Activity peaked at 50 micrograms/ml and was lost if 0.2% IL-1 antisera were added to the cultures.

Aberrant regulation of IL-1 expression in macrophages from young autoimmune-prone mice.

IL-1 is a multifunctional, immunoregulatory polypeptide produced by many cell types. Because activated macrophages are a major source of IL-1 and have also been implicated in the pathogenesis of autoimmune disease, we investigated the regulation of IL-1 expression in several autoimmune-prone strains of mice. Peritoneal macrophages derived from the autoimmune-prone strains MRL/lpr, MRL/+, NZB, and NZB/W F1, as well as NZW, displayed transient expression of IL-1 in contrast to the stable expression characteristic of control normal strains including A. Thy, A/J, B10, B10.A, B10.D2, C57BL/6, BALB/c, and C3H/HeN. The down-regulation of IL-1 by macrophages from the autoimmune-prone mice was not attributable to inherently defective signal transduction because macrophages from both the normal and autoimmune-prone strains displayed substantial initial levels of cell-associated and secreted IL-1. However, during the first 2 to 3 days in culture, macrophages from autoimmune-prone mice became progressively refractory to both induction and maintenance of IL-1, a pattern that correlated with changes in the levels of IL-1 alpha and beta mRNA. The progressive reduction in IL-1 expression by macrophages from these autoimmune-prone strains was not due to a reduction in general metabolism or viability, because expression of cell surface antigens, including MHC class I and II Ag and LFA-1, was comparable to that of control macrophages. Because IL-1 plays a critical role in the homeostasis of a variety of cell lineages, defective expression, and maintenance of IL-1 (and perhaps other cytokines) by macrophages from the autoimmune-prone strains may contribute to the immune dysregulation that develops in these mice. Alternatively, cytokine dysregulation might not contribute directly to disease, but rather reflect a more basic defect related to specific signal transducing or gene regulatory pathways.

CAMP differentially regulates expression of mRNA encoding IL-1 alpha and IL-1 beta in murine peritoneal macrophages.

The influence of PGE2 and the consequent rise in intracellular cAMP on LPS-induced IL-1 alpha and IL-1 beta mRNA levels has been examined in murine peritoneal macrophages. As has been previously reported, neither PGE2 nor dibutyryl cAMP modulated the levels of LPS-induced IL-1 alpha mRNA. In contrast, the levels of IL-1 beta mRNA were markedly potentiated (greater than 10 fold) under the same experimental conditions. This effect was due to elevation of intracellular cAMP because other agents known to elevate cAMP levels (e.g., cholera toxin, forskolin, 1-isomethyl-3-isobutylxanthine) had the same selective effect on IL-1 beta mRNA levels. PGE2 and dBcAMP not only potentiated LPS-induced IL-1 beta mRNA levels but were also able to stimulate modest accumulation of IL-1 beta mRNA in the absence of LPS. Although measurement of IL-1 activity by bioassay suggested that dBcAMP and PGE2 could suppress LPS-induced IL-1 expression, levels of IL-1 protein, determined by radioreceptor assay, were markedly elevated. cAMP did not appreciably alter the stability of either IL-1 alpha or IL-1 beta mRNA. Instead dBcAMP independently stimulated the transcriptional activity of the IL-1 beta gene. In concert the results demonstrate that cAMP can modulate the response of mononuclear phagocytes to LPS in a complex pattern; gene expression may be unaltered, suppressed, or potentiated and will thereby affect both the quality and magnitude of the ensuing inflammatory response.

Interleukin-4 inhibits interluekin-1 synthesis by a posttranscriptional mechanism

T cell derived cytokines, interleukin-2 and interferon-gamma, are known to upregulate the interleukin-1 synthesis of monocyte/macrophages. The effect of a third T cell derived cytokine, interleukin-4, was now investigated. Human peripheral blood monocytes were activated with bacterial lipopolysaccharide in the presence or absence of interleukin-4 and the production of biologically active interleukin-1 was quantitated both from the culture supernatants and from the monocyte lysates using the mouse thymocyte assay. Interleukin-4 had a clear dose-dependent suppressive effect on the interleukin-1 synthesis. IL-1 alpha and IL-1 beta mRNA levels, quantitated by Northern blot analysis, were equally high in the lipopolysaccharide plus interleukin-4 supplemented cultures and in the cultures with lipopolysaccharide alone, suggesting that the effect of interleukin-4 in the interleukin-1 biosynthesis is post-transcriptional.

Differential effects of in vitro mycoplasma infection on interleukin-1 alpha and beta mRNA expression in U937 and A431 cells.

Cultured cells depend on cytokine mediators for sustained growth and maintenance and are routinely employed in bioassays to detect and measure minute changes in biological mediators, e.g. the interferons and interleukins. We evaluated the effects of mycoplasma infection on the steady-state mRNA levels of two cytokines IL-1 alpha and beta. Noninfected human squamous carcinoma cell line A431 expressed constitutively IL-1 alpha and beta mRNA. In contrast freshly isolated peripheral blood mononuclear cells and the monocytic cell line U937 expressed abundant IL-1 mRNA only after the appropriate stimulation. Peripheral blood mononuclear cells and U937 steady-state IL-1 beta mRNA levels were considerably greater than IL-1 alpha mRNA levels, whereas nearly equivalent high levels of IL-1 alpha and beta mRNA were detected in A431 cells. Mycoplasma infection of cultured A431 cells reduced the steady-state levels of IL-1 alpha and beta mRNA. This effect was nonspecific for A431 cells as actin mRNA steady-state levels showed similar decreases to mycoplasma contamination. However, this response was cell specific since mycoplasma-free and contaminated U937 cells differed little in IL-1 mRNA expression. These results show that the response to mycoplasma infection is at least partly cell-type dependent.