In vitro analysis of B lymphocyte function in uraemia.

We have investigated the immune responses in vitro of uraemic patients undergoing regular haemodialysis or continuous ambulatory peritoneal dialysis. Twenty-five healthy subjects were also studied as controls. In uraemic patients, the number of T and B lymphocytes were within the normal range, but proliferative responses to phytohaemagglutinin (PHA) were impaired. Spontaneous immunoglobulin plaque forming cell (PFC) responses by peripheral blood mononuclear cells (PBMC) from uraemic patients were significantly lower than those of healthy subjects. The PFC response of uraemic PBMC to the T cell independent polyclonal B cell activator (PBA) Epstein-Barr virus (EBV) was comparable to the response of the healthy subjects, indicating that uraemic B cells are still capable of synthesizing immunoglobulin. Pokeweed mitogen (PWM) induced PFC responses of uraemic PBMC were also normal, whereas the response to another T cell dependent B cell activator, Staphylococcus aureus Cowan I (SAC), was very low. Addition of indomethacin to PWM- and SAC-activated cultures of uraemic PBMC enhanced the PFC response to SAC, but had little effect on the PWM response. As full differentiation of B cells in response to SAC depends on helper T cells, we conclude that a defect in T lymphocyte function accounts for the reduced spontaneous and SAC induced production of immunoglobulin by uraemic PBMC. This defect may be mediated by an indomethacin-sensitive mechanism.     

Production of tumor necrosis factor alpha in human leukocytes stimulated by Cryptococcus neoformans.

Tumor necrosis factor alpha (TNF-alpha) is a key mediator of inflammation and may promote human immunodeficiency virus replication in latently infected cells. Since cryptococcosis often is associated with aberrations in the host inflammatory response and occurs preferentially in persons with AIDS, we defined the conditions under which human leukocytes produce TNF-alpha when stimulated by Cryptococcus neoformans. Peripheral blood mononuclear cells (PBMC) produced comparable amounts of TNF-alpha following stimulation with C. neoformans and lipopolysaccharide. Detectable TNF-alpha release in response to C. neoformans occurred only when fungi with small-sized capsules were used and complement-sufficient serum was added. Fractionation of PBMC established that monocytes were the predominant source of TNF-alpha. TNF-alpha gene expression and release occurred significantly later in PBMC stimulated with C. neoformans than in PBMC stimulated with LPS. C. neoformans was also a potent inducer of TNF-alpha from freshly isolated bronchoalveolar macrophages (BAM). Upon in vitro culture, BAM and monocytes bound greater numbers of fungal cells, yet their capacity to produce TNF-alpha following cryptococcal stimulation declined by 74 to 100%. However, this decline was reversed if the BAM and monocytes were cultured with gamma interferon. These data establish that C. neoformans can potently stimulate TNF-alpha release from human leukocytes. However, several variables profoundly affected the amount of TNF-alpha released, including the type of leukocyte and its state of activation, the size of the cryptococcal capsule, and the availability of opsonins.     

Effect of pro-inflammatory/anti-inflammatory agents on cytokine secretion by peripheral blood mononuclear cells in rheumatoid arthritis and systemic lupus erythematosus

We studied a well-selected population of patients with active rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) without immunosuppressive therapy. Control and patient peripheral blood mononuclear cells (PBMC) were incubated with IL-1beta, IL-10, TGF-beta or LPS for 20 h and the in vitro basal and stimulated secretions of IL-6, TNF-alpha, IL-1beta and IL-1ra were measured by ELISA. We found that in the SLE patients the basal secretion of IL-6 was significantly lower and that of IL-1ra significantly higher than in control subjects, while in the RA group the basal IL-1ra secretion was higher than in healthy subjects. SLE and RA PBMC responded to LPS and IL-1beta reaching higher cytokine secretion values than controls. The in vitro response of SLE and RA PBMC to TGFbeta was normal, while that to IL-10 was defective: IL-10 was able to stimulate the production of IL-6 and IL-1ra in PBMC from normal subjects, but it was unable to enhance IL-6 secretion in RA cells and it was also completely ineffective in inducing IL-1ra secretion in both SLE and RA PBMC. Our work add new data useful for the evaluation of IL-10 and IL-1ra as therapeutic agents in rheumatic diseases.     

Effects of gamma interferon on release of tumor necrosis factor alpha from lipopolysaccharide-tolerant human monocyte-derived macrophages.

After an initial stimulation of human monocyte-derived macrophages with bacterial lipopolysaccharide (LPS), which produces substantial release of tumor necrosis factor-alpha (TNF-alpha), a subsequent exposure to LPS results in about an order-of-magnitude reduction in the levels of TNF-alpha released. We have shown that macrophages which have been stimulated with LPS and then maintained in culture without LPS for as long as 2 weeks do not regain their original capacity to secrete TNF-alpha upon a second LPS challenge. After 2 to 4 days in adherent culture, monocyte-derived macrophages which were not pretreated with LPS also experience a measurable decline in their capacity to release TNF-alpha in response to an initial LPS stimulation. When compared with these previously nonstimulated cells, however, the levels of TNF-alpha released by LPS-pretreated cells in response to a second LPS challenge decline by over 90% after 8 to 9 days in culture. Unstimulated cells spontaneously release barely detectable levels of TNF-alpha. In contrast to the release of TNF-alpha, unstimulated cells release significant levels of prostaglandin E2 continuously over time, and these levels are variably increased by no more than a factor of two in response to a single LPS stimulation. Prostaglandin E2 levels released by LPS-pretreated cells in response to a second LPS stimulation are much closer to the levels released by unstimulated cells. We have also demonstrated that gamma interferon (IFN-gamma) enhances TNF-alpha release from LPS-stimulated macrophages but not from phorbol myristate acetate-stimulated cells. Addition of IFN-gamma to macrophages either during the initial stimulation or during a second stimulation with LPS enhances levels of TNF-alpha released after the second LPS challenge. The greatest enhancement is observed when IFN-gamma is added during both exposures to LPS, but addition of IFN-gamma during only the initial LPS stimulation still results in marked enhancement of TNF-alpha release in response to a second stimulation with LPS 24 h later. If an interval of 2 days of culture in medium alone separates the first and second 24-h LPS stimulations, IFN-gamma enhances TNF-alpha release only when it is included during the second LPS exposure, indicating that, unlike the persistence of endotoxin tolerance, enhancement of TNF-alpha release by IFN-gamma is transient.     

Developmental regulation of the cytokine repertoire in human macrophages: IL-1, IL-6, TNF-alpha, and M-CSF

We were interested in the dependence of constitutive and stimulated cytokine secretion on the stage of macrophage (MAC) differentiation in vitro. Elutriation-purified blood MO were cultured up to 28 days and their secretory repertoire was analyzed under adherence conditions at various culture stages. For each of the cytokines tested, interleukin (IL)-1 beta, IL-6, tumor necrosis factor (TNF)-alpha, and macrophage colony-stimulating factor (M-CSF), a different pattern of regulation was observed. During the initial phase of maturation (up to day 7 in culture) within which the characteristics of normal MO to MAC transformation are achieved, M-CSF was the only cytokine to be secreted constitutively. From the LPS-dependent cytokines, IL-1 beta and IL-6 were downregulated whereas TNF-alpha levels increased severalfold. For the release of IL-1 beta, IL-6, and TNF-alpha a synergistic effect of interferon-gamma (IFN-g) and lipopolysaccharide (LPS) was observed. M-CSF release increased until day 7 in culture with LPS being stimulatory for this particular cytokine only during the first days of differentiation. Upon further cultivation of MAC up to 28 days, LPS-induced IL-1 beta levels remained very low, but IL-6 levels increased again reaching that of blood MO, and TNF-alpha continued to rise reaching levels up to 30-fold higher than in blood MO. M-CSF secretion stayed high with LPS even suppressing constitutive secretion. Long-term cultured MAC started to release IL-6 and TNF-alpha also in the absence of a stimulus and, furthermore, became responsive to IFN-g alone. Our data show that the release of each cytokine investigated is differently regulated during maturation. These results document the functional plasticity of human MAC and emphasize the impact that MO to MAC differentiation may have in vivo.     

Heat shock and 5-azacytidine inhibit nitric oxide synthesis and tumor necrosis factor-alpha secretion in activated macrophages

To elucidate the role of stress response during macrophage activation, the effects of heat shock and the amino acid analog, 5-azacytidine on nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-alpha) secretion, and heat shock protein (HSP) synthesis have been studied in murine peritoneal macrophages (C57BL/6). Heat shock (1 hr at 43 degrees C) or 5-azacytidine markedly inhibited the release of NO into the medium from interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-stimulated macrophages. Although heat shock significantly decreased TNF-alpha secretion only at the initiation stage of macrophage stimulation, 5-azacytidine treatment resulted in a more prolonged reduction in the secretion of TNF-alpha. When heat-shocked cells were stimulated with IFN-gamma plus LPS under normal culture conditions at 37 degrees C, the heat shock-induced inhibition of NO release reversed progressively with increasing recovery time. Although the total amount of cellular HSP72 measured by Western blot increased time-dependently over 7 hr, newly synthesized HSP72 measured by [35S]methionine incorporation was evident only after 1 and 3 hr of recovery time after heat shock treatment. At these time points, the lowest nitrite accumulation and TNF-alpha secretion into the medium was evident. It is concluded that signaling pathways related to newly synthesized HSP such as HSP72 are implicated in the down regulation of NO synthesis and TNF-alpha secretion in macrophages.     

Direct influence of mild hypothermia on cytokine expression and release in cultures of human peripheral blood mononuclear cells.

Hypothermia is associated with elevated frequency of infectious complications. Dysfunction of the immune response caused by hypothermia has been demonstrated in both clinical and animal studies, but it still remains unclear to what extent immunocompetent cells are directly influenced by hypothermia. To estimate the direct influence of mild hypothermia on cytokine expression and release by human peripheral blood mononuclear cells (PBMC), primary cultures of PBMC were incubated at 34 degrees C or 32 degrees C activated by lipopolysaccharide (LPS), phytohemagglutinin (PHA), or tumor necrosis factor-alpha (TNF-alpha). The cytokine gene expression was evaluated by RT-PCR. Release of interleukin-2 (IL-2), IL-6, IL-10, and TNF-alpha was measured by ELISA. Mild hyperthermia significantly impaired IL-2 gene expression in PHA-stimulated cultures of PBMC and decreased IL-2 release in all variants of cultures. Secretion of IL-6, IL-10, and TNF-alpha was decreased in hypothermic cultures of PBMC stimulated with the T lymphocyte activator PHA. Slight suppression of IL-10 secretion was observed also in TNF-alpha-stimulated hypothermic cultures of PBMC. TNF-alpha release increased slightly in mild hypothermia control cultures. Our data demonstrate that the direct influence of hypothermia on cytokine expression and release from PBMC is not uniform. Reduction of IL-2 production might play a crucial role in the impairment of immune response in hypothermia.     

Role of IFN-gamma and alpha in IL 1 synthesis and secretion of in vitro differentiated human macrophages: a comparative study

Human blood monocytes (Mo) cultured in vitro differentiate to macrophages (Mx) and lose the capacity to secrete interleukin 1 (IL 1) in response to endotoxin (LPS). Incubation of Mo with interferon gamma or alpha (IFN-gamma or IFN-alpha) prevented this loss of IL 1 secretory potential during the first 24 h of culture. However, there were marked differences between the two interferons if culture period was extended beyond 24 h. Incubation of Mo with IFN-gamma for 48 or 72 h induced IL 1 release in response to LPS in all the donors without exception. In contrast, 48-h incubation of Mo with IFN-alpha alpha caused IL 1 secretion (in response to LPS) in only a minority of donors, while 72-h incubation resulted in very little or no IL 1 release in all the individuals tested. Moreover, only IFN-gamma had the capacity to reinduce IL 1 secretory potential in Mx which had lost the capacity to secrete IL 1 during previous culture. These and other results suggest that IFN-alpha differs from IFN-gamma in being: a less potent IL 1 inducer, ineffective in maintaining IL 1 secretory capacity of fresh Mo for more than 48-72 h, completely unable to reinduce IL 1 secretory potential in culture-derived Mx. Thus, the two species of IFN appear to have a markedly different role in IL 1 synthesis and secretion.     

Induction of tumour necrosis factor-alpha during haemodialysis. Influence of the membrane type.

Some of the secondary clinical effects induced by long-term haemodialysis in patients with end-stage renal failure have been related to an increased production of interleukin-1 (IL-1). We investigated the role of another cytokine which shares a number of biological properties with IL-1, tumour necrosis factor-alpha (TNF-alpha). In long-term haemodialysed patients, we found at the beginning of the dialysis increased plasma TNF-alpha levels and enhanced monocyte capacity to produce TNF-alpha spontaneously ex vivo. Non-haemodialysed uraemic patients also presented increased plasma TNF-alpha levels. During dialysis with cellulose acetate (CA) or polysulphone (PS) membranes, plasma TNF-alpha levels and the spontaneous and lipopolysaccharide-induced production of TNF-alpha by monocytes remained at predialysis levels. In contrast, when cuprophane membranes were used, there was a significant increase in plasma TNF-alpha levels and in both spontaneous (10-fold) and lipopolysaccharide-induced (seven-fold) ex vivo TNF-alpha production by monocytes. These results suggest that monocytes are stimulated during haemodialysis with the poorly biocompatible cuprophane membrane.     

Bacterial lipopolysaccharide induces release of tumor necrosis factor-alpha from bovine peripheral blood monocytes and alveolar macrophages in vitro

In this study, we demonstrate that freshly adherent bovine monocytes release tumor necrosis factor-alpha (TNF-alpha) in response to stimulation with bacterial lipopolysaccharide (LPS). TNF-alpha was detected using actinomycin D-treated WEHI-164 murine fibrosarcoma cells as targets in an 18 hr cytotoxicity assay. Doses of LPS from 20 ng/ml to 20 micrograms/ml were capable of inducing bovine TNF-alpha. The kinetics of TNF-alpha release from bovine monocytes demonstrated peak levels of cytotoxic activity at 1-3 hr post-LPS treatment, with a subsequent decline to background levels by 18 hr post-LPS treatment. A monoclonal antibody that neutralizes recombinant human TNF-alpha (rHuTNF-alpha) significantly reduced the cytotoxicity of LPS-stimulated bovine monocyte culture supernatants. Size exclusion high-performance liquid chromatography (HPLC) analysis of LPS-stimulated monocyte and alveolar macrophage culture supernatants resulted in a molecular weight elution profile similar to that of recombinant human TNF-alpha. These elution profiles are consistent with the presence of multimers of TNF-alpha. This is believed to be the first report of the in vitro production of bovine TNF-alpha.     

Echinococcus multilocularis metacestodes modulate cellular cytokine and chemokine release by peripheral blood mononuclear cells in alveolar echinococcosis patients

Infection with the cestode Echinococcus multilocularis causes human alveolar echinococcosis (AE), a life-threatening disease affecting primarily the liver. Despite the severity of AE, clinical symptoms often develop only many years after infection, which suggests that E. multilocularis has developed mechanisms which depress anti-parasite immune response, thus favouring immune evasion. In this study we examined the production of cytokines, chemokines and the expression of CD molecules on peripheral blood mononuclear cells (PBMC) from AE patients and healthy controls in response to E. multilocularis metacestode culture supernatant, viable E. multilocularis vesicles and E. multilocularis vesicle fluid antigen in vitro. After 48 h of co-culture, E. multilocularis metacestode culture supernatant and E. multilocularis vesicles depressed the release of the proinflammatory cytokine interleukin (IL)-12 by PBMC. This effect was dose-dependent and a suppression of tumour necrosis factor (TNF)-alpha and IL-12 was observed even when PBMC were activated with lipopolysaccharide (LPS). Comparing proinflammatory cytokine release by AE patients and controls showed that the release of IL-12 and TNF-alpha was reduced in AE patients, which was accompanied by an increased number of CD4+ CD25+ cells and a reduced release of the Th2 type chemokine CCL17 (thymus and activation regulated chemokine, TARC), suggesting an anti-inflammatory response to E. multilocularis metacestode in AE patients. Instead the production of interferon (IFN)-gamma and the expression of CD28 on CD4+ T cells were increased in PBMC from AE patients when compared to controls. This was accompanied by a higher release of the Th2-type chemokine CCL22 (macrophage derived chemokine, MDC) supporting that E. multilocularis also generates proinflammatory immune responses. These results indicate that E. multilocularis antigens modulated both regulatory and inflammatory Th1 and Th2 cytokines and chemokines. Such a mixed profile might be required for limiting parasite growth but also for reducing periparasitic tissue and organ damage in the host.     

Differences in cytokine secretion by intestinal mononuclear cells, peripheral blood monocytes and alveolar macrophages from HIV-infected patients.

Mononuclear cells of the lamina propria (LpMNC), isolated from endoscopically taken biopsies of the large bowel from AIDS patients, were analysed for their ability to secrete tumour necrosis factor-alpha (TNF-alpha), IL-1 beta and IL-6. Stimulation of LpMNC from normal controls with pokeweed mitogen (PWM) led to a time- and dose-dependent enhancement of TNF-alpha, IL-1 beta and IL-6 secretion. In contrast, PWM stimulation of LpMNC from AIDS patients resulted in only a small increase in TNF-alpha release. Constitutive secretion of IL-1 beta and IL-6 in these patients was already increased to the concentration range of stimulated cells from normal controls and could not be further increased, probably due to maximal in vivo stimulation. Secretion of TNF-alpha, IL-1 beta and IL-6 by peripheral blood monocytes (PBM) and alveolar macrophages from AIDS patients was elevated with or without stimulation compared with normal controls. Obviously, the regulation of TNF-alpha secretion is dependent on the microenvironment. Since it is known that interferon-gamma (IFN-gamma) may induce the production of TNF-alpha, the secretion of this cytokine was examined. Release of IFN-gamma was constitutively and under stimulation lowered in LpMNC from AIDS patients compared with normal controls. Addition of IFN-gamma to LpMNC did not result in enhanced TNF-alpha secretion. Our data indicate a defective function of intestinal mononuclear cells in AIDS patients as shown by the diminished TNF-alpha secretion.     

Immunoglobulin D enhances the release of tumor necrosis factor-alpha, and interleukin-1 beta as well as interleukin-1 receptor antagonist from human mononuclear cells.

Immunoglobulin D (IgD) is normally present in only low concentrations in serum. In the hyper-IgD and periodic fever syndrome (HIDS), however, serum levels exceed 140 mg/l. This syndrome is further characterized by recurrent inflammatory febrile attacks together with an acute phase response and appearance of cytokines in the circulation. The role of IgD in the pathogenesis of HIDS and its relation to the increased cytokine concentrations is unclear. Therefore, we tested whether IgD, IgG and alpha 1-acid glycoprotein (AGP) isolated from human serum influence the synthesis of interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), and IL-1ra, as measured by specific radioimmunoassays, in human peripheral blood mononuclear cells (PBMC). Incubation of PBMC with IgD and AGP for 24 hr led to increased release of IL-1 beta, TNF-alpha, and IL-lra. The magnitude of stimulation of IgD exceeded that of AGP; the effect by IgD was dose-dependent and showed a 30-fold (TNF-alpha) to almost 150-fold (IL-1 beta) increase at the highest concentration (50 mg/l), while AGP (750 micrograms/ml) only increased the cytokine secretion fourfold (TNF-alpha) to almost 30-fold (IL-1 beta). The effect of IgD on IL-1ra was less dramatic but a fivefold increase was observed at 50 mg/l compared with a 2.5-fold increase with AGP. IgD potentiated the effect of lipopolysaccharide (LPS) on secretion of both IL-1 beta and TNF-alpha, although the effect was most apparent for TNF-alpha. Apart from inducing IL-1ra synthesis, IgG did not influence cytokine release in human PBMC. These data indicate that IgD is a potent inducer of TNF-alpha, IL-1 beta and IL-1ra and thus may contribute to the pathogenesis of HIDS.     

Effect of Pro-inflammatory/Anti-inflammatory Agents on Cytokine Secretion by Peripheral Blood Mononuclear Cells in Rheumatoid Arthritis and Systemic Lupus Erythematosus

We studied a well-selected population of patients with active rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) without immunosuppressive therapy. Control and patient peripheral blood mononuclear cells (PBMC) were incubated with IL-1 &#103, IL-10, TGF- &#103 or LPS for 20 h and the in vitro basal and stimulated secretions of IL-6, TNF- &#102, IL-1 &#103 and IL-1ra were measured by ELISA. We found that in the SLE patients the basal secretion of IL-6 was significantly lower and that of IL-1ra significantly higher than in control subjects, while in the RA group the basal IL-1ra secretion was higher than in healthy subjects. SLE and RA PBMC responded to LPS and IL-1 &#103 reaching higher cytokine secretion values than controls. The in vitro response of SLE and RA PBMC to TGF &#103 was normal, while that to IL-10 was defective: IL-10 was able to stimulate the production of IL-6 and IL-1ra in PBMC from normal subjects, but it was unable to enhance IL-6 secretion in RA cells and it was also completely ineffective in inducing IL-1ra secretion in both SLE and RA PBMC. Our work add new data useful for the evaluation of IL-10 and IL-1ra as therapeutic agents in rheumatic diseases.     

Lymphocyte transformation in human plasma without addition of synthetic medium. A study of immune function in patients with chronic uraemia or diabetes mellitus.

The in vitro response of lymphocytes obtained from normal subjects, uraemic patients on haemodialysis and diabetic patients was studied using cultures containing either medium plus plasma (medium cultures) or plasma alone (plasma cultures). The study demonstrated that plasma alone can adequately support lymphocyte transformation induced by nonspecific mitogens (PHA and PWM) and allogeneic lymphocytes in mixed lymphocyte culture (MLC) reaction. This investigation further confirms our previously reported findings that uraemic patients undergoing haemodialysis have a normal lymphocyte response in MLC and to PHA and PWM. Plasma cultures give results similar to conventional medium cultures in subjects where lymphocyte transformation is normal. The lymphocyte hyporactivity observed in diabetics is, however, better shown in the plasma cultures. The suppressed response of the diabetic patient's lymphocytes to PHA and PWM both in the presence of autologous and normal AB plasma suggests intrinsic lymphocyte dysfunction as the explanation for impaired immune function. Plasma cultures may provide a better in vitro system for the evaluation of immune function in certain groups of patients where it is desirable to distinguish between intrinsic abnormalities of lymphocyte function and the effect of humoral immunosuppressive factors.     

Peripheral blood mononuclear cells from patients with rheumatoid arthritis spontaneously secrete vascular endothelial growth factor (VEGF): specific up-regulation by tumour necrosis factor-alpha (TNF-alpha) in synovial fluid.

This study was designed to investigate VEGF production from peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) compared with healthy controls and to identify the predominant cellular source in PBMC isolated from RA patients. The regulation of PBMC VEGF production by cytokines and synovial fluid (SF) was studied. PBMC were isolated from RA patients and healthy controls and stimulated with lipopolysaccharide (LPS), IL-1beta, IL-4, IL-6, IL-8, IL-10, TNF-alpha and transforming growth factor-beta (TGF-beta) isoforms for varying time points up to 72 h at 37 degrees C/5% CO2. The effect of SF on VEGF secretion by PBMC was also studied. Supernatant VEGF levels were measured using a flt-1 receptor capture ELISA. RA patients had significantly higher spontaneous production of VEGF compared with controls, and monocytes were identified as the predominant cellular source. RA PBMC VEGF production was up-regulated by TGF-beta isoforms and TNF-alpha and down-regulated by IL-4 and IL-10, with no effect observed with IL-1beta, IL-6 and IL-8. Antibody blocking experiments confirmed that TNF-alpha and not TGF-beta isoforms in SF increased VEGF secretion by RA PBMC. These results emphasize the importance of monocytes as a source of VEGF in the pathophysiology of RA. Several cytokines known to be present in SF can modulate the level of VEGF secretion, but the predominant effect of SF in VEGF up-regulation is shown to be dependent on TNF-alpha.     

Influence of enteric parasitism on hormone-regulated pancreatic secretion in dogs.

The objective of this study was to test the hypothesis that enteric parasites affect pancreatic secretion in their host. Pancreatic bicarbonate and protein outputs were studied in dogs with gastric and pancreatic fistulas to determine the secretory response to exogenously administered secretin and cholecystokinin and to intraduodenal stimulation with hydrochloric acid and sodium oleate to release endogenous hormones. Bicarbonate and protein concentrations in pancreatic juice were measured prior to infection with Trichinella spiralis and at various periods during primary and secondary infections. Dose-related increases in secretory activity were observed in uninfected hosts in response to all stimuli. Infected dogs responded like controls to exogenous hormones, but showed reduced secretion under duodenal stimulation during the 1st wk of primary infection. This altered response returned to normal 3 wk after primary infection and did not occur following secondary infection. Results support the conclusion that reduced pancreatic secretion is associated with enteric parasitism and is due to a defect in hormone release or in the supply of hormone available for release.     

CCL20/macrophage inflammatory protein 3alpha and tumor necrosis factor alpha production by primary uterine epithelial cells in response to treatment with lipopolysaccharide or Pam3Cys

Having previously shown that CCL20/macrophage inflammatory protein 3alpha and tumor necrosis factor alpha (TNF-alpha) are released by polarized primary rat uterine epithelial cells (UEC) in response to Escherichia coli but not to Lactobacillus rhamnosus, we sought to determine if epithelial cells are responsive to pathogen-associated molecular patterns (PAMP), including lipopolysaccharide (LPS), lipoteichoic acid (LTA), and Pam(3)Cys, a bacterial lipoprotein analog. Epithelial cells were grown to confluence on Nunc cell culture inserts prior to apical treatment with PAMPs. In response to LPS, LTA, and Pam(3)Cys (EMC Microcollection GmbH, Tubingen, Germany), CCL20 levels increased (4- to 10-fold) while PAMPs caused increased TNF-alpha (1- to 4-fold) in the medium collected after 24 h of incubation. Both apical and basolateral secretion of CCL20 and TNF-alpha increased in response to PAMPs, but treatments had no effect on cell viability and integrity, as measured by transepithelial resistance. Time course studies of CCL20 and TNF-alpha release in response to Pam(3)Cys and LPS indicated that CCL20 release peaked between 2 and 4 h after treatment, whereas TNF-alpha release was gradual over the length of the incubation. Freeze-thaw and cell lysis experiments, along with actinomycin D studies, suggested that CCL20 and TNF-alpha are synthesized in response to PAMP stimulation. Taken together, these studies demonstrate that E. coli and selected PAMPs have direct effects on the production of CCL20 and TNF-alpha without affecting cell integrity. Since CCL20 is known to be both chemotactic and antimicrobial, the increase in apical and basolateral release by UEC in response to PAMPs suggests a new mechanism of innate immune protection in the female reproductive tract.     

Production of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) in patients with AIDS. Enhanced level of TNF-alpha is related to a higher cytotoxic activity

We measured simultaneously circulating and cell-generated TNF-alpha and IL-1 after lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells (PBMC) by radioimmunoassay (RIA) in HIV-infected individuals at different stages of infection, classified according to CDC classification. TNF-alpha production, both in vitro and endogenous in sera, remained at the normal level in group II patients but was significantly increased in most patients in group IV (P less than 0.05). Most patients of group II and IV displayed normal level of IL-1 in their sera, whereas the level of this monokine generated in vitro was significantly reduced in both groups (P less than 0.05). The cytotoxic effect of factor(s) secreted by PBMC from HIV-infected individuals was evaluated towards a fibroblast cell line L929. The higher titre of cytotoxicity was directly related to a higher production of TNF-alpha by the cells from group IV patients and the effect could be removed by pre-absorption with anti-TNF-alpha monoclonal antibody.     

Enhanced proinflammatory response to endotoxin after priming of macrophages with lead ions

Exposure to lead ions strongly enhances the susceptibility of rodents to endotoxin shock and parasitical infections. Macrophages play a key role during the immune response to lipopolysaccharide (LPS) and during the defense against parasites and might be a target of lead. In the present study, bone marrow-derived macrophages (BMMphi) pretreated with lead chloride prior to stimulation with LPS were analyzed for their release of immune mediators. Lead-pretreated cells released up to tenfold increased amounts of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-12, and prostaglandin E(2) (PGE(2)) but less IL-10 compared with controls. These effects were paralleled by enhanced mRNA levels and were dependent on the duration of lead pretreatment. Inhibition of protein kinase C or of protein synthesis during the priming phase blocked the lead-induced increase of TNF-alpha and IL-6 release. In conclusion, lead ions prime BMMphi for enhanced proinflammatory cytokine secretion in response to LPS, likely by activation of protein kinase C and subsequent synthesis of an unidentified mediator.