Interleukin-1 regulates the proliferation of leukocytes in human corneal cell-peripheral blood leukocyte cocultures.

We have documented the inability of human corneal epithelial-like cells to suppress proliferation of peripheral blood leukocytes (PBLs) induced by allogeneic PBLs in a mixed leukocyte reaction (MLR). Instead, enhanced proliferation of PBLs, albeit small, was consistently noted as indicated by uptake of radiolabeled thymidine. Maximum proliferation of PBLs was detected when a mixed leukocyte reaction (MLR) was conducted in the presence of corneal cells. High levels of interleukin-1 beta (IL-1 beta) were found during MLR irrespective of the presence of corneal cells. High levels of IL-1 beta correlated well with observed synergistic stimulation of PBL proliferation by corneal cells and stimulating allogeneic PBLs. In PBL-corneal cell cocultures, PBLs produced IL-1 beta; corneal cells contributed large amounts of prostaglandin E2 (PGE2). Although indomethacin completely blocked prostaglandin E2 production, it did not significantly alter the results. Our data show that PBLs and corneal cells can reciprocate each other's presence, and, under appropriate conditions, corneal cells can deliver at least one signal to enhance rather than suppress antigen-driven PBL proliferation. Our data suggest a role for immunoregulatory cytokines and prostanoids such as IL-1 beta and PGE2 in these interactions.

Cytokines cause cultured retinal pigment epithelial cells to secrete metalloproteinases and to contract collagen gels.

PURPOSE: Because retinal pigment epithelial cells in epiretinal membranes remodel and contract their surrounding extracellular matrix, investigations were performed to determine if these cells can produce matrix metalloproteinases and contract collagen gels in vitro in the presence of serum or cytokines. METHODS: Cells were grown on collagen gels and their production of metalloproteinases was measured using zymography. RESULTS: Cells grown in a three-dimensional collagen gel culture system produce several latent metalloproteinases that are secreted into the gel and the surrounding medium. These include molecules of 49, 56, 66, and 100 kD. In addition, an enzyme that is probably the active form of the 66 kD enzyme is present. When interleukin 1 beta is added to the cultures, latent 49 kD and 100 kD gelatinase production is greatly stimulated and an active form of both enzymes is also observed in the medium. In contrast, transforming growth factor beta has no stimulatory effect. The cells contract the collagen gel but this is small without cytokines; however, contraction is greatly enhanced in the presence of serum or interleukin 1 beta plus transforming growth factor beta. Contraction is unlikely to be the result of metalloproteinase action on the underlying extracellular matrix because complete inhibition of these enzymes has little effect. CONCLUSIONS: These results show that cytokines can cause cultured retinal pigment epithelial cells to produce metalloproteinases that can, when activated, degrade most collagens and other structural molecules in extracellular matrix. In addition, they can stimulate the contraction of extracellular matrix constituents but there is not a simple causal relationship between matrix remodeling and contraction.

Regulation of mitogen-driven lymphoreticular cell activation by human corneal cells and interleukin-1.

Keratin-positive fibroblast-like epithelial cells (FLE), isolated from human corneo-scleral-conjunctival rims, were shown to inhibit mitogen-driven (concanavalin A) DNA synthesis by murine thymocytes and splenocytes [lymphoreticular cells (LRC)]. The effect exerted by live cells in culture and by their supernatants was caused by factors active across species barriers. Paraformaldehyde-fixed or irradiated cells also suppressed mitogen-induced thymocyte DNA synthesis, but their supernatants manifested no such activity. Interaction between FLE cells and LRC in the presence of the mitogen resulted in suppressed cellular activation as evidenced by significantly lowered tetrazolium salt (MTT) reduction in murine thymocytes and splenocytes, suggesting reduced mitochondrial activity. The suppressive effect was seen with live and paraformaldehyde-fixed FLE cells. There was a good correlation between MTT assays and [3H]thymidine uptake experiments. Suppression of MTT reduction in murine thymocytes and splenocytes by intact FLE cells could be reversed by the addition of interleukin-1 (IL-1). Indomethacin prevented FLE-conditioned medium-induced suppression but failed to relieve suppression by whole FLE cells. Thus, suppression of LRC function by FLE cells and their secretions appeared to operate by different mechanisms. One mechanism related to prostaglandins present in FLE cell-conditioned medium, whereas another mechanism appeared to involve cell-membrane-associated factor(s). The findings not only provide additional information on the capability of corneal cells to regulate lymphoreticular cells but suggest an important role for IL-1 in the regulation of LRC function and corneal inflammation and immunity.

Cholylglycine measured in serum by RIA and interleukin-1 beta determined by ELISA in differentiating viral hepatitis from chemical liver injury.

Serum bile acids have been shown to serve as useful indicators of liver disease. We have confirmed these findings and added an analysis of interleukin-1 beta (IL-1 beta) profiles to further differentiate viral hepatitis from toxic liver damage associated with exposure to vinyl chloride (VC) or trinitrotoluene (TNT). The frequency of elevated cholylglycine (CG) was 100%, 75%, and 37.5% in viral hepatitis, VC- and TNT-linked liver injury patients, respectively. The mean levels, expressed in micrograms/dL, were 578, 507, 142, and 65 in hepatitis B, hepatitis non-A non-B, VC and TNT liver injury patients, respectively. Thus, the CG test could detect viral hepatitis and, VC liver injury, and (less frequently) liver injury associated with exposure to TNT. The mean level of IL-1 beta in patients with hepatitis type B was 424 pg/mL and hepatitis non A non B was 384 pg/mL compared with a mean of 33-40 pg/mL in those with VC or TNT-linked liver disease. The IL-1 beta detection test proved further to be an important distinguishing parameter as it was 100% positive in patients with viral hepatitis but only 12.5% to 25% positive in patients with VC/TNT-induced liver damage.

Interleukin-1 beta induces interleukin-1 alpha messenger ribonucleic acid expression in primary cultures of Leydig cells.

Previously, we have reported that interleukin-1 (IL-1) can modulate Leydig cell steroidogenesis. Recently, IL-1-like material has been shown to be present in the testis; however, the cellular source of this material remains unclear. In the present study we found that human recombinant IL-1 beta (1-100 ng/ml) caused dose-dependent increases in IL-1 alpha mRNA expression in Leydig cells. Similar to that reported in other tissues, IL-1 alpha mRNA from Leydig cells is mainly 2.2 kilobases. IL-1 alpha mRNA expression in Leydig cells was detectable as early as 2 h after the addition of IL-1 beta (10 ng/ml) and persisted for up to 24 h. Lipopolysaccharide also stimulated IL-1 alpha mRNA expression in these cells, but phorbol ester had no effect. Our results indicate that Leydig cells are a potential source of IL-1, which has both autocrine and paracrine effects.

Interleukin-2 is a potent inhibitor of Leydig cell steroidogenesis.

Interstitial tissue of the testis consists of Leydig cells, macrophages, lymphocytes, plasma cells, mast cells and fibroblasts. Previously we have reported that interleukin-1 (IL-1) inhibits Leydig cell androgen production. In the present study, the effect of IL-2 was investigated. Leydig cells (10(5) cells/ml) from adult Sprague-Dawley rats were cultured with or without IL-2 for 24 h. After medium changes, human CG (hCG), 8-bromo-cAMP, or forskolin was added with or without IL-2. Cultures were continued for an additional 24 h, and testosterone and cAMP levels were measured. IL-2 up to 100 U/ml had no effect on basal testosterone production. hCG-stimulated testosterone formation was inhibited in a dose-dependent manner by the addition of IL-2. IL-2 in a concentration of 100 U/ml decreased hCG-induced testosterone formation from 49.6 +/- 3.6 ng/ml (mean +/- SE) to 8.5 +/- 4.2 ng/ml. The hCG dose-response curve was shifted to the right by the addition of IL-2. Maximal testosterone production in response to hCG was reduced 40% in the presence of IL-2 (50 U/ml) without alteration of median effective dose (ED50). IL-2 also inhibited hCG-induced cAMP formation and 8-bromo cAMP- and forskolin-stimulated testosterone production. However, IL-2 did not alter the binding of [125I]hCG to purified Leydig cells. Furthermore, IL-2 significantly inhibited the conversion of 20-OH-cholesterol, 22-OH-cholesterol, pregnenolone, progesterone, 17 alpha-hydroxypregnenolone, and 17 alpha-hydroxyprogesterone to testosterone but did not alter the conversion of dehydroepiandrosterone and androstenedione to testosterone. Our results suggest that a T cell growth factor, IL-2, is a potent inhibitor of steroidogenesis. IL-2 may play a paracrine role in modulating Leydig cell function.

Differential effects of recombinant interleukin-1 alpha and beta on Leydig cell function.

Previously we have reported that human chorionic gonadotropin(hCG)-stimulated testosterone biosynthesis was markedly inhibited by purified natural human interleukin-1 (IL-1). In the present study we evaluated the effects of human and murine recombinant IL-1 (rIL-1) on Leydig cell steroidogenesis in primary culture. Human rIL-1 beta caused a dose-dependent inhibition of hCG-, 8-bromo cyclic AMP-, and forskolin-induced testosterone formation. In contrast, human rIL-1 alpha was considerably less potent. When the effects of the cytokines were corrected for their biological potencies, human rIL-1 beta and murine rIL-1 alpha were still more effective than human rIL-1 alpha in inhibiting testosterone production (at least 100-fold more potent). Thus, even though IL-1 alpha and IL-1 beta bind to the same receptors on T cells, Leydig cells exhibit differential sensitivity in response to rIL-1 alpha and rIL-1 beta which is partly species dependent.

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.

Interferon-gamma, Staphylococcus aureus, and lipopolysaccharide/silica enhance interleukin-1 beta production by human corneal cells.

Cultures derived from human corneo-scleral rims remaining after a central corneal button had been removed for transplantation, revealed two types of cells on light microscopy: One with typical epithelial morphology and the other resembling fibroblasts. Both cell types contained keratin filaments in early passage and were therefore considered epithelial in nature. The fibroblast-like cells were designated fibroblast-like epithelial cells (FLE) while the typical epithelial cells were referred to as E-type. Both E and FLE cells constitutively produced an IL-1-like factor as determined by thymocyte proliferation assay and IL-2 induction in EL-4 lymphoma cells. Moreover, the supernatants from these cells potentiated concanavalin A (Con A)-primed mitochondrial oxidative metabolism in thymocytes, as indicated by the tetrazolium salt reduction assay (MTT) and this effect was significantly neutralized with monoclonal anti-IL-1 beta. The release of biologically active IL-1 beta by the FLE cells is another characteristic (in addition to the presence of keratin) distinguishing these cells from fibroblasts which do not release biologically active IL-1 beta. Using an ELISA, specific for IL-1 beta, there was clear cut evidence for increased production of this cytokine by FLE cells in response to human recombinant gamma-interferon (IFN-gamma), Staphylococcus aureus, and lipopolysaccharide (LPS) in combination with silica (LPS/silica). Time studies with IFN-gamma and LPS/silica demonstrated that enhanced production was time dependent and that IL-1 beta was primarily cell associated. The results indicate that human corneal E- and FLE-type cells can produce and release IL-1 and that FLE cells can be induced by inflammatory mediators to increase production of IL-1 beta.

Interleukin-1 inhibits Leydig cell steroidogenesis in primary culture.

Inflammation and infection induce an acute phase response. The response is characterized by fever and production of interleukin-1 (IL-1). In the present study we evaluated the effects of interleukin-1 on Leydig cell function in primary culture. hCG-stimulated testosterone formation was markedly reduced by IL-1, with an ED50 of 1 U/ml. Basal testosterone production was slightly enhanced in the presence of low concentrations of IL-1, while high concentrations of IL-1 inhibited testosterone formation. Significant inhibition of hCG-stimulated testosterone formation was noted as early as 8 h after the addition of IL-1. IL-1 also inhibited hCG-stimulated cAMP formation, as well as 8-bromo-cAMP- and forskolin-stimulated testosterone synthesis. Furthermore, LH binding to Leydig cells was reduced by human IL-1. The inhibitory effects of IL-1 were reversed only partially by the addition of a cyclooxygenase inhibitor, indomethacin (0.1 mM), even though prostaglandin E2 formation was completely blocked. This indicates that the observed effects of IL-1 are not completely mediated by increased PGE2 formation. The present study suggests that IL-1 is a potent modulator of Leydig cell steroidogenesis. Decreased testosterone formation may modulate the immune response and contribute to the catabolic changes occurring during infection.

Corneal epithelial cells produce thromboxane in response to interleukin 1 (IL-1).

Synthesis of thromboxane, a product of arachidonic acid formed via the cyclooxygenase pathway, was studied in rabbit corneal epithelial cells (SIRC cell line) under resting conditions and under the influence of interleukin 1 (IL-1). IL-1 potentiated the production of thromboxane 3-10-fold in a dose-dependent manner. This finding assumes added significance in view of the previous observations that the same cells are capable of producing IL-1. Thus, the corneal epithelial cells may be viewed in this context as an autocrine cell producing two biologically active substances which can serve as mediators of inflammation, one of which can augment the production of the other.

Phylogenetic studies on lymphokines. Fish lymphocytes respond to human IL-1 and epithelial cells produce an IL-1 like factor.

The results obtained indicate, that catfish peripheral blood lymphocytes recognize and respond to human IL-1. The second part of this report is dealing with a substance produced by carp epidermal cells with functional similarities to mammalian IL-1.

Fish lymphocytes respond to human IL-1.

IL-1 is known to cross species barriers in that IL-1 of one mammalian species can interact with cells of other mammalian species. The work described herein demonstrates that IL-1 can cross phylogenetic classes as IL-1 derived from human peripheral blood lymphocytes (PBL) potentiated the proliferative response of catfish lymphocytes to the T cell mitogen Concanavalin A (ConA). Catfish PBL were separated by density gradient centrifugation and used in blastogenic transformation assays. IL-1 had relatively little direct stimulatory effect but with suboptimal amounts of ConA there was a significant augmentation of the proliferative response. The specificity of the response was determined by neutralization experiments in which anti-human IL-1 antibody significantly diminished the reaction of catfish lymphocytes to IL-1.

Resistance of helper T lymphocytes to cyclophosphamide.

The effect of cyclophosphamide (Cy) on helper T lymphocytes using an adoptive transfer approach in athymic nude mice was investigated. The results indicated that Cy, at a dose (100 mg/kg) which virtually abolished anti-sheep erythrocyte (SRBC) antibody plaque forming cell (PFC) response of Balb/c mice, did not alter significantly the capacity of their splenic T cells to restore the anti-SRBC PFC response of nude mice. This resistance of T helper cells was observed in unimmunized mice and in mice injected with SRBC two days prior to Cy administration. It has been concluded that both resting and antigen stimulated T helper cells responsible for reconstituting anti-SRBC response of nude mice are resistant to Cy.

Enhancement of natural resistance to influenza virus in lipopolysaccharide-responsive and -nonresponsive mice by Propionibacterium acnes.

Lipopolysaccharide-responsive C3H/HeN mice were rendered resistant to a mouse-adapted strain of influenza (Aichi, H(3)N(2)) virus when Propionibacterium acnes was given either intranasally or intraperitoneally several days before virus infection. The time of P. acnes treatment was important since no protection was demonstrated when this agent was given either on the same day as or several days after virus challenge. In contrast, lipopolysaccharide-nonresponsive C3H/HeJ mice were not protected when P. acnes was administered intranasally at any time before infection; however, protection was demonstrated when P. acnes was given by the intraperitoneal route. Depending on the route of inoculation, P. acnes induced several distinctive immunological responses in the lungs of both C3H/HeN and C3H/HeJ mice. Intranasal inoculation was more effective in activating pulmonary macrophages in C3H/HeN than in C3H/HeJ mice. In contrast, intraperitoneal inoculation activated pulmonary natural killer cells in both mouse lines but did not activate pulmonary macrophages.