Interleukin-15 plays an essential role in the pathogenesis of autoimmune diabetes in the NOD mouse.

AIMS/HYPOTHESIS: IL-15, induced by innate immune stimuli, promotes rheumatoid arthritis and inflammatory bowel disease. However, its role in autoimmune type 1 diabetes is unclear. Our aim is to define the role of IL-15 in the pathogenesis of diabetes in the NOD mouse model. METHODS: We generated NOD.Il15(-/-) mice expressing a polyclonal repertoire of T cell antigen receptor (TCR) or a transgenic TCR and monitored diabetes onset and insulitis. NOD Scid.Il15(-/-) (full name NOD.CB17-Prkdc (scid)/NCrCrl) and NOD Scid.gamma (full name NOD.Cg-Prkdc(scid) Il2rg ( tm1Wjl )/SzJ) mice were used to distinguish the requirement for IL-15 signalling in CD8(+) T cells and antigen-presenting cells (APCs) to induce disease. We examined the effect of blocking IL-15 signalling on diabetes onset in NOD mice. RESULTS: At 7 months of age, more than 75% of the NOD Il15(-/-) female mice remained diabetes free compared with only 30% in the control group. Diabetes incidence was also decreased in 8.3-NOD (full name NOD Cg-Tg[TcraTcrbNY8.3]-1Pesa/DvsJ).Il15(-/-) mice expressing a highly pathogenic transgenic TCR on CD8(+) T cells. Adoptive transfer of splenocytes from diabetic NOD and 8.3-NOD donors induced disease in NOD Scid recipients but not in NOD Scid.Il15(-/-) or NOD Scid.gamma mice. Transient blockade of IL-15 signalling at the onset of insulitis prevented diabetes in NOD mice. CONCLUSIONS/INTERPRETATION: Our results show that IL-15 is needed for the initial activation of diabetogenic CD8(+) T cells as well as for sustaining the diabetogenic potential of antigen-stimulated cells, acting on both CD8(+) T cells and on APCs. Our findings demonstrate a critical role for IL-15 in the pathogenesis of autoimmune diabetes and suggest that IL-15 is a promising therapeutic target.

Cysteinyl-leukotrienes induce vascular endothelial growth factor production in human monocytes and bronchial smooth muscle cells.

BACKGROUND: Cysteinyl leukotrienes (cysLTs) are suggested to be implicated in the process of airway remodelling in asthma. OBJECTIVE: We investigated the potential for cysLTs to modulate vascular endothelial growth factor (VEGF) expression, a growth factor involved in the angiogenesis of airway remodelling. METHODS: VEGF mRNA and protein were quantified by real-time PCR and ELISA, respectively. VEGF promoter activation was assessed using luciferase gene-tagged promoter constructs. RESULTS: We found that LTD(4) induction of VEGF in human monocytes and bronchial smooth muscle cells is cysLT1 dependent. Stimulation of HEK293 cells stably expressing cysLT1 or cysLT2 with cysLTs showed a concentration-dependent activation of the VEGF promoter and a time-dependent increase in VEGF mRNA and protein. For the cysLT1-mediated response, mutations of hypoxia-induced factor-1 (HIF-1) sites failed to reduce cysLT-induced VEGF promoter activation and 5' deletions showed that the proximal region containing one AP-1 and four specificity protein 1 (Sp1) sites was necessary. Pretreatment with inhibitors of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not p38, and an overexpression of dominant negative forms of c-Jun, c-Fos or Ras suggested the implication of mitogen-activated protein kinases and AP-1. Mutation of the AP-1-binding element failed to prevent VEGF transactivation suggesting that AP-1 might not act directly on the promoter. Moreover, inhibition of Sp1-dependent transcription by mithramycin completely inhibited VEGF promoter transactivation and VEGF mRNA expression by LTD(4) . Finally, mutations of Sp1 binding elements prevented VEGF promoter transactivation. CONCLUSION AND CLINICAL RELEVANCE: Our data indicate for the first time that cysLTs can transcriptionally activate VEGF production via cysLT1 receptors, with the involvement of JNK, ERK, the AP-1 complex and Sp1. These findings suggest that cysLTs may be important in the angiogenic process of airway remodelling and potentially provide a previously unknown benefit of using cysLT1 receptor antagonists in the prevention or treatment of airway remodelling in asthma.

Transforming growth factor-beta1 in asthmatic airway smooth muscle enlargement: is fibroblast growth factor-2 required?

Enlargement of airway smooth muscle (ASM) tissue around the bronchi/bronchioles is a histopathological signature of asthmatic airway remodelling and has been suggested to play a critical role in the increased lung resistance and airway hyperresponsiveness seen in asthmatic patients. The pleiotropic cytokine, TGF-beta1, is believed to contribute to several aspects of asthmatic airway remodelling and is known to influence the growth of many cell types. Increased TGF-beta1 expression/signalling and ASM growth have been shown to occur concurrently in animal models of asthma. Abundant studies further substantiate this association by showing that therapeutic strategies that reduce or prevent TGF-beta1 overexpression/signalling lead to a parallel decrease or prevention of ASM enlargement. Finally, recent findings have supported a direct link of causality between TGF-beta1 overexpression/signalling and the overgrowth of ASM tissue. To follow-up on these in vivo studies, many investigators have pursued detailed investigation of ASM in cell culture conditions, assessing the direct role of TGF-beta1 on cellular proliferation and/or hypertrophy. Inconsistencies among the in vitro studies suggest that the effect of TGF-beta1 on ASM cell proliferation/hypertrophy is contextual. A hypothesis focusing on fibroblast growth factor-2 is presented at the end of this review, which could potentially reconcile the apparent discrepancy between the conflicting in vitro findings with the consistent in vivo finding that TGF-beta1 is required for ASM enlargement in asthma.

Leukotriene D4-induced, epithelial cell-derived transforming growth factor beta1 in human bronchial smooth muscle cell proliferation.

BACKGROUND: Cysteinyl-leukotrienes (cys-LTs) orchestrate many pathognomonic features of asthma in animal models of allergic airway inflammation, including bronchial smooth muscle cell (BSMC) hyperplasia. However, because cys-LTs alone do not induce mitogenesis in monocultures of human BSMC, the effect observed in vivo seemingly involves indirect mechanisms, which are still undefined. OBJECTIVE: This study aims to investigate the regulatory role of leukotriene (LT)D(4) on TGF-beta1 expression in airway epithelial cells and the consequence of this interplay on BSMC proliferation. METHODS: HEK293 cells stably transfected with cys-LT receptor 1 (CysLT1) (293LT1) were stimulated with LTD(4) and TGF-beta1 mRNA and protein expression was measured using Northern blot and ELISA, respectively. Conditioned medium (CM) harvested from LTD(4)-treated cells was then assayed for its proliferative effect on primary human BSMC. TGF-beta1 mRNA expression was also determined in tumoural type II pneumocytes A549 and in normal human bronchial epithelial cells (NHBE) following LTD(4) stimulation. RESULTS: The results demonstrated that LTD(4)-induced TGF-beta1 mRNA production in a time- and concentration-dependent manner in 293LT1. TGF-beta1 secretion was also up-regulated and CM from LTD(4)-treated 293LT1 was shown to increase BSMC proliferation in a TGF-beta1-dependent manner. The increased expression of TGF-beta1 mRNA by LTD(4) also occured in A549 and NHBE cells via a CysLT1-dependent mechanism. CONCLUSION: In conclusion, elevated expression of cys-LTs in asthmatic airways might contribute to BSMC hyperplasia and concomitant clinical features of asthma such as airway hyperresponsiveness via a paracrine loop involving TGF-beta1 production by airway epithelial cells.

Expression of platelet-activating factor receptor in human carotid atherosclerotic plaques: relevance to progression of atherosclerosis.

BACKGROUND: Human monocyte-derived macrophages synthesize numerous proinflammatory and prothrombotic substances, including lipid mediators, such as platelet-activating factor (PAF), which may play a major role in the onset and perpetuation of atherosclerotic lesions. In addition, both monocytes and macrophages express PAF receptors (PAF-R). The expression of PAF-R is transcriptionally downregulated by oxidized LDL in in vitro primary cultures of monocyte/macrophages. In this study, we evaluated the expression of PAF-R in human carotid plaque tissue, in foam cells isolated from human carotid plaques, and in primary cultures of umbilical smooth muscle cells (SMCs). METHODS AND RESULTS: We show that PAF-R was expressed at low levels in foam cells compared with monocyte/macrophages in plaques, as assessed by immunohistochemical staining and in situ hybridization. In addition, low levels of mRNA were also detected by RT-PCR in isolated human carotid foam cells. A prominent finding of our study was the demonstration that contractile SMCs were positive for PAF-R, and its mRNA was extracted from primary cultures of umbilical SMCs. CONCLUSIONS: As macrophages loose their inflammatory phenotype on transformation into foam cells, they may equally loose their capacity of defense against aggression. We postulate that the diminished expression of PAF-R may be deleterious in the context of plaque formation and progression. The observation that arterial SMCs of contractile phenotype express PAF-R opens new avenues concerning the migration of these cells from media to intima and atherosclerotic plaque formation.

Fucose-activated killer cells. I. Enhanced TNF-alpha mRNA accumulation and protein production.

Our previous studies have shown that the monosaccharide alpha-L-fucose significantly enhances the cytolytic capacity of peripheral blood mononuclear leukocytes (PBMLs). To examine possible mechanisms through which fucose affects cytolytic activity, we studied the production of cytokines after alpha-L-fucose stimulation. In this report, we show that fucose induced a minor but significant augmentation of production of interleukin-2 (IL-2), but anti-IL-2 antibodies did not completely inhibit fucose-activated cytolysis. Fucose induced significantly higher secretion of TNF-alpha by both lymphocytes and monocytes. The nature of the lytic molecule detected in the TNF bioassay was verified with specific neutralizing antibodies. In addition, fucose induced the accumulation of TNF-alpha mRNA in a time-dependent manner with a peak at 8 h and a return to baseline values at 20 h after stimulation. In vitro nuclear transcription assays determined that fucose augmented the rate of transcription of the TNF-alpha gene, and inhibition of de novo transcription with actinomycin D indicated that the turnover rate of the TNF-alpha mRNA was not affected by fucose stimulation. We also determined that fucose did not modulate the mRNA expression of the pore-forming protein, a major lytic protein involved in lymphocyte cytotoxicity. Specific neutralizing antibodies indicated that TNF-alpha was not an effector molecule in fucose-activated killing of K562 or Raji target cells but that this cytokine had an essential role in the induction of the augmented killing by alpha-L-fucose.

Differentiation-dependent modulation of TNF production by PAF in human HL-60 myeloid leukemia cells.

Platelet-activating factor (PAF) can augment tumor necrosis factor (TNF) production by human monocytes in a bimodal manner, with two peaks of activation at picomolar and micromolar concentrations. These peaks are partially associated with monocyte subsets presenting different characteristics in terms of size, density, phenotypic markers, and [Ca2+]i mobilization responses. In the present study, we used the human promyelocytic leukemia cell line HL-60, at various times during differentiation with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] toward the monocyte lineage, in order to study the relation of cell differentiation to responsiveness to PAF in terms of cytokine production. TNF production was induced by pretreatment with interferon gamma for 24 h and treatment with muramyl dipeptide. Although detectable TNF was produced by 4 day-differentiated cells, no effect was seen with PAF (10(-16)-10(-6) M) at this or earlier stages. In contrast, 5 day-differentiated cells had a comparable baseline production of TNF but responded with a 2.5-fold increase to PAF with a single peak, maximal at 10(-8) M. Moreover, 6 day- or 7 day-differentiated HL-60 cells showed a further increase in TNF production in response to PAF, and the response was bimodal, similar to that of the less dense subset of monocytes, with peaks at 10(-14) and 10(-7) M PAF. In parallel, undifferentiated HL-60 failed to respond to PAF in terms of [Ca2+]i mobilization. The earliest responsiveness to PAF (10(-7) M) was observed by 4 days of treatment with 1,25(OH)2D3, and by day 7 the response to PAF became bimodal (10(-14) and 10(-7) M). These results indicate that myeloid cells acquire, during maturation toward the monocyte lineage, a progressive responsiveness to PAF in terms of [Ca2+]i mobilization and enhanced cytokine production, and they suggest that the heterogeneity in responses to PAF observed in normal monocytes may be related to their stage of differentiation or maturation.

Augmented expression of platelet-activating factor receptor gene by TNF-alpha through transcriptional activation in human monocytes.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine produced by activated monocytes and often associated with platelet-activating factor (PAF) during the pathogenesis of many inflammatory and infectious diseases. PAFR is a G-protein-coupled receptor constitutively expressed on monocytes. TNF-alpha (100-400 U/mL) significantly increased PAFR mRNA expression in human monocytes. This increase was seen after 1 h of stimulation and persisted up to 24 h. Actinomycin D pretreatment studies revealed a transcriptional increase in PAFR gene expression without effect on mRNA half-life. [3H]WEB 2086 binding studies showed a significant (43%) increase in specific binding sites in 24-h-treated cells without change in receptor affinity. Increased interleukin-6 production in response to PAF was also found in 24-h TNF-alpha-pretreated monocytes. These observations provide new evidence for TNF-alpha and PAF interactions in human monocytes during inflammatory processes through up-regulation of PAFR expression by TNF-alpha.

Decreased p21 levels in anti-sense ras transfectants augments NK sensitivity.

We have shown that the oncogenic EJ-ras gene, under the control of a metallothionein-I (Mt) promoter, can be induced to cause an increased susceptibility of transfected 10T1/2 fibroblasts to cytolysis mediated by natural killer (NK) cells. This effect may be specific to the ras gene family, since other oncogenes that we have tested here (src) and elsewhere (myc) do not show this effect. We have now examined the effect of modulating the level of p21 in both a positive or negative manner. The level of p21 ras was decreased by two independent mechanisms. First Zn2+ was removed from Mt-EJ-ras transformed cells. In the second approach we transfected 10T1/2 cells with a Mt-anti-sense c-H-ras construct which reduced p21 expression, slowed the growth rate and altered the morphology of 10T1/2 cells when induced with Zn. Surprisingly, the decrease in p21 ras levels by both approaches caused a marked increase in NK susceptibility (NKS) which was equivalent to that observed when the p21 ras levels were increased either by inducing EJ-ras or removing Zn2+ from Mt-anti-sense c-H-ras containing cells. The kinetics of induction of NK sensitivity due to decreasing normal p21 ras levels was identical to that observed for increasing mutated p21 ras levels. Peak enhancement of NKS was observed 24 hr after ras perturbation. These results suggest that either a positive or negative change in the steady-state level of p21 ras is sufficient to induce NK sensitivity, and NK sensitivity is not inextricably linked to cellular transformation by the ras gene.

Role of the Cys90, Cys95 and Cys173 residues in the structure and function of the human platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent phospholipid mediator which binds to a specific, high affinity receptor of the G protein-coupled receptor family. In the present report, we show that ligand binding to the PAF receptor is sensitive to the reducing agent dithiothreitol (DTT), suggesting the involvement of disulfide linkages in the proper PAF receptor conformation. Substitutions of Cys90, Cys95 and Cys173 to Ala or Ser demonstrated that these cysteine residues are critical for normal cell surface expression of the PAF receptor protein and ligand binding to the receptor. The Cys90 and Cys173 mutant receptors did not display any specific ligand binding, were not expressed on the cell surface but were found in the intracellular compartment. The Cys95 mutants showed specific binding and were able to stimulate low levels of inositol phosphate (IP) production. These mutants were expressed at low density on the cell surface and showed high expression intracellularly. Our results suggest that the structure and function of the PAF receptor require the conserved Cys90 and Cys173 to form a disulfide bond. Moreover, Cys95 also appears to be necessary, possibly by establishing a disulfide linkage with an as yet unidentified Cys residue. All three residues appear essential for the proper folding and surface expression of the PAF receptor protein.

The in vivo clearance of Ha-ras transformants by natural killer cells.

The experiments in this study were designed to test the hypothesis that natural killer (NK) cells play a role in host surveillance against early neoplastic changes in the malignant process. C3H 10T1/2 mouse fibroblasts were transfected with a pSV2-neo plasmid vector which contains EJ, the mutated c-Ha-ras, regulated by its own promoter. Control cells were transfected with pSV2-neo alone and did not contain the ras gene. Oncogene-transfected cells were compared with control cells for lung colony formation following tail vein injection into C3H mice. Intravenous injection of ras-transfected 10T1/2 cells induced marked lung colony formation in vivo, whereas C3H 10T1/2 parental lines or 10T1/2 cells transfected with pSV2-neo alone induced no lung colonies in C3H mice. The colonising potential of ras transfectants could be decreased by augmentation of NK activity by injection of polyinosinic cytidylic acid and increased by depletion of NK effectors with anti-asialo GM1. Experiments with beige mice demonstrated that the mortality of syngeneic, NK-deficient C3H-bg/bg mice injected with ras tranfectants was significantly greater than similarly treated NK-normal C3H(-)+/bg littermate controls. The results support the view that NK cells are capable in vivo of recognizing early defined stages in the neoplastic process initiated by oncogenes.

Changes in apoptosis of human polymorphonuclear granulocytes with aging.

Many alterations with aging occur at the cellular and organic levels in the immune system ultimately leading to a decrease in the immune response. Our aim in the present work was to study apoptosis of polymorphonuclear granulocytes (PMN) with aging under various stimulations since apoptosis might play an important role in several pathologies encountered with aging. The PMN of healthy young (20-25 years) and elderly (65-85 years) subjects were examined after 24 h of sterile culture with and without stimulation. The stimulating agents included: phorbol myristate acetate (PMA), hydrogen peroxide (H2O2), N-formyl-methionyl-leucyl phenylalanine (FMLP), granulocyte-macrophage colony stimulating factor (GM-CSF), reduced glutathione (GSH), lipopolysaccharide (LPS) and interleukin 2 (IL-2). Apoptosis was assessed by traditional staining of the plates, by flow cytometric staining and DNA gel electrophoresis. It was found that without stimulation the susceptibility of PMN to apoptosis was slightly increased with aging. Under various stimulations, such as PMA. H2O2, apoptosis was almost 100%, while the treatment by FMLP, oxLDL and GSH did not change its extent in PMN obtained either from young or elderly subjects. Marked age-related changes were observed in the extent of apoptosis under stimulation with GM-CSF, IL-2 and LPS. These agents were able to significantly prevent apoptosis in PMN of young subjects, while only the GM-CSF was able to slightly modulate it in neutrophils of elderly subjects. From these results, we suggest that changes in apoptosis of PMN with aging could play a role in the increased incidence of certain immune system related pathologies of aging, such as cancer, infections and autoimmune disorders.

High efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation.

Endothelial cells derived from the human umbilical vein (HUVEC) are used to study the mechanisms involved in EC response to various stimuli as well as to investigate the basis of pathological conditions of the vascular system such as altered endothelium permeability, tumor-induced angiogenesis, atherosclerosis and leukocyte extravasation in chronic inflammatory responses. However, investigations of gene involvement related to these conditions have progressed slowly because of the difficulty of transfecting HUVEC with high efficiency. Whereas several technical approaches have been described, they usually result in low levels of transfected cells or they require several steps or sophisticated instrumentation. We describe here a straightforward protocol of transfection of freshly isolated HUVEC that is based on the simple technique of electroporation. Efficiencies of gene transfection greater than 40% were routinely obtained by using a combination of optimized conditions of HUVEC isolation, composition of the electroporation medium and homogeneity of the plasmids. The protocol has been applied to the functional transient transfection of functional genes in HUVEC as illustrated in the case of the cDNA encoding GFP, protein kinase C (alpha and epsilon isotypes) and beta-galactosidase.

PAF activation of a voltage-gated R-type Ca2+ channel in human and canine aortic endothelial cells.

By the use of fura-2 and digital imaging techniques, [K]o depolarization or PAF (10(-9) M) were shown to induce a sustained increase of [Ca]i in human or canine single aortic vascular endothelial cells (VEC) that was insensitive to nifedipine but sensitive to (-)-PN200-110 or to lowering of [Ca]o. The PAF-induced effect on [Ca]i was blocked by the PAF receptor antagonist, WEB2170. Our results suggest that [K]o depolarization and PAF increase [Ca]i via the activation of R-type Ca2+ channels.

Suppressor cells in the human maternal-fetal relationship.

The mixed lymphocyte culture (MLC) of maternal and newborn (cord) cells is significantly weaker than that of father-newborn and control-newborn cultures. This hyporeactivity was found not to be due to an impaired function or tolerance of either the maternal or neonatal cells. We investigated the possibility that a specific, in vivo-induced suppressor cell was active in the diminished maternal-newborn reaction. Suppressor cells were found to be active in both the stimulating and responding populations in the unidirectional MLC. The removal of TG cells from the responding (maternal or newborn) population resulted in an increase of reactivity specific for the corresponding stimulating population (newborn or maternal). The suppressor activity within the stimulating population was carried out by a radiosensitive cell, which did not require proliferation to exert its effect. We suggest that the observed hyporeactivity of maternal-newborn mixed lymphocyte cultures is due to the modulation of the reaction by specific, in vivo-induced suppressor cells.

Interleukin 6 production by mononuclear phagocytes can be stimulated by leukotrienes.

Leukotriene B4 is a potent lipid mediator of inflammation, and some of its bioactivities may involve inflammatory cytokines. Human monocytes, cultured in the presence of graded concentrations of LTB4, were significantly stimulated in their production of IL6. Nanomolar concentrations of the mediator were optimal for stimulation of IL6 production, which was already significant at 6 hours. LTC4 showed a similar, albeit lower activity. In addition to stimulating IL6 protein production, LTB4 also augmented IL6 mRNA accumulation, which was maximal at 1 hour. Furthermore, LTB4-treated monocytes contained increased amounts of nuclear protein capable of binding to potential transcriptional promoter regions of the IL6 gene. These data suggest that leukotrienes may modulate the production of IL6 and indicate some underlying mechanisms which may be involved.

Increased micronuclei frequencies in couples with reproductive failure.

A higher incidence of chromosomal instability in the infertile population is widely recognized. An increased level of micronuclei has been shown to be a marker of chromosome damage. Therefore, micronuclei frequencies were assessed in cytokinesis-blocked lymphocytes of 130 patients (65 couples) with idiopathic infertility or with two or more spontaneous abortions, and 30 healthy fertile donors (15 couples). The frequency of micronucleated cells in the cohort with reproductive failure and healthy controls averaged 14.95+/-6.04 per 1000 and 10.60 +/-2.57 per 1000 (P<0.0001), respectively. When micronuclei frequency sums in particular couples (male + female) were analyzed in the same order, identical statistical significance was reached (P<0.0001). We found no effect of age or sex on micronuclei frequency. In summary, the cytokinesis-blocked micronuclei assay revealed increased micronucleus frequency in couples with infertility or two or more spontaneous abortions, suggesting a possible role of chromosomal instability in reproductive failure.

Glucose tolerance and erythrocyte insulin receptors in Friedreich's ataxia.

Detailed in vivo and in vitro studies of glucose and insulin metabolism in Friedreich's ataxia patients and unaffected family members have further defined the extent of the abnormalities in carbohydrate metabolism. The high incidence of glucose intolerance and a hyperinsulinemic response to a glucose challenge in a high percentage of Friedreich's ataxia patients has been confirmed. An increased incidence of glucose intolerance among heterozygotes is suggested, while the siblings show a more normal distribution of diabetes and a nearly normal insulin response to the glucose tolerance test. Human growth hormone patterns are normal for all groups. Preliminary studies of insulin binding to erythrocytes suggest a difference in the binding characteristics among diabetic Friedreich's ataxia patients, while the binding in the non-diabetic Friedreich's ataxia group is similar to that of non-diabetic controls. Results from a small group of non-diabetic siblings suggest a normal insulin binding, while a tendency toward increased binding at low insulin concentrations among diabetic family members is noted.

Platelet-activating factor stimulates interleukin-6 production by human endothelial cells and synergizes with tumor necrosis factor for enhanced production of granulocyte-macrophage colony stimulating factor.

The interaction between human endothelial cells (EC) and leukocytes during inflammation is in part mediated through the release of soluble factors. Since platelet-activating factor (PAF) is a potent mediator of inflammatory responses, we investigated the potential of PAF to modulate IL-6 and GM-CSF production by EC. Exposure of these cells to PAF resulted in a concentration-dependent increase in IL-6 production, with a maximum at 10(-10) M PAF. Sequential incubation of EC with PAF and TNF alpha resulted in a synergistic increase of IL-6 production. This effect was specific for PAF since it was prevented by preincubation with the PAF receptor antagonist, WEB 2086. Northern blot analysis revealed enhanced IL-6 mRNA expression in PAF-treated EC. However, the synergy observed in protein synthesis between PAF and TNF alpha was not reflected in IL-6 mRNA accumulation, suggesting a post-translational modulation. Pretreatment of EC with the protein synthesis inhibitor cycloheximide before their exposure to PAF resulted, after washout of the cycloheximide, in a markedly augmented production of IL-6, suggesting a synergy between augmented IL-6 mRNA accumulation by PAF and IL-6 mRNA superinduction by cycloheximide. GM-CSF production by EC was also stimulated by the combined effects of PAF and TNF alpha, but PAF alone did not affect GM-CSF production. Taken together, our data suggest that PAF can stimulate EC to synthesize cytokines, including IL-6 and GM-CSF, which may contribute to local and, possibly, systemic responses during inflammation.

Natural killer and lectin-dependent cytotoxic activities of Kurloff cells: target cell selectivity, conjugate formation, and Ca++ dependency.

Kurloff cells may represent a major component of NK cell activity in the guinea pig. We have pursued to characterize the mechanism of their action. Using murine target cells, we found Kurloff cell cytotoxicity to be selective for the NK-sensitive YAC-1 target cell, with minimal activity against the NK-resistant P815 target cell. In the presence of PHA, but not ConA, cytotoxicity was markedly augmented against both YAC-1 and P815. While effector-target conjugate formation was observed with YAC-1 cells but not P815 cells in control cultures, it was augmented with both target cell types in cultures with PHA. Pretreatment alone with PHA was ineffective, however. NK cell activity of Kurloff cells was dependent on extracellular Ca++ and entry of Ca++ into the effector cells, as demonstrated by abrogation of cytotoxicity when extracellular Ca++ was chelated with EDTA or EGTA, or following treatment with the Ca++ channel blockers verapamil and diltiazem. Furthermore, inhibition of PKC by H7 resulted in significant reduction of Kurloff cell-mediated NK activity, while pretreatment of effector cells with the PKC activator TPA enhanced NK activity. Kurloff cells could also be stimulated to produce serine esterases by contact with target cells or treatment with phorbol ester and ionophore. Finally, a majority of Kurloff cells, identified by the monoclonal antibody 14D1, reacted with the human NK cell marker CD56. Taken together, these data suggest that Kurloff cells have NK-like characteristics and activity, with target cell selectivity, and that their lytic mechanisms involve influx of extracellular Ca++, PKC activation and serine esterase production.