A multidonor ELISPOT study of IL-1β, IL-2, IL-4, IL-6, IL-13, IFN-γ and TNF-α release by cryopreserved human peripheral blood mononuclear cells

Utilization of cryopreserved peripheral blood mononuclear cells (PBMCs), rather than fresh ones collected from the same donor on different dates, overcomes the variability in sensitivity of these cells to activation agents. To understand the effect of cryopreservation, frozen PBMCs from eight healthy donors were studied to release T(H)1 or T(H)2 cytokines including IL- 1 beta, IL-2, IL-4, IL-6, IL-13, TNF-alpha and IFN-gamma using ELISPOT assay. The number of spot-forming cells (SFC) was determined using three concentrations of PBMCs (5 x 10(6), 5 x 10(5) and 5 x 10(4) cells/ml). PBMCs from all eight donors were found to retain their functional capacity to release T(H)1 or T(H)2 cytokines after freezing and thawing. When PBMCs were taken in concentrations 5 x 10(6) or 5 x 10(5) cells/ml, the density of IL-1 beta-, IL-2-, IL-6- and TNF-alpha-related spots in a well for most of the donors appeared to be overly high, making SFC quantification either difficult or impossible. To the contrary, PBMCs in concentration 5 x 10(4) produced distinct and quantifiable spots. The density of spots related to IL-4 and IL-13 release appeared to be optimal for SFC quantification when PBMCs were taken in concentration 5 x 10(6) whereas in 5 x 10(5) cells/ml the spot density was very low and absent in 5 x 10(4) cells/ml concentration group. No relationship between release levels of different cytokines was found, except IFN-gamma and IL-2 cytokine indicating that cryopreserved PBMCs with a high IFN-gamma response will likely have a high IL-2 response as well. Our results indicate that a release level of one cytokine may not be reliably predicted by knowing the level of the other. This implies that it is necessary to test cryopreserved PBMCs in a broad range of concentrations to determine one, which will be optimal for producing distinct and quantifiable spots.     

IL-1β TNF-α, and IL-2 in Peritoneal Fluid and Macrophage-Conditioned Media of Women With Endometriosis

PROBLEM : The presence of various cytokines in human peritoneal fluid has been incompletely evaluated. Changes in cytokine levels may be related to the development of endometriosis, infertility, and activation of peritoneal macrophages. This study assesses levels of IL-1β, IL-2, and TNF-α in peritoneal fluid and macrophage conditioned media of women with endometriosis. METHOD : Peritoneal fluid was collected from 51 women at the time of diagnostic or operative laparoscopy for benign gynecologic disease. Peritoneal macrophages were isolated, cultured for 24 h, and the culture media collected. IL-1β, IL-2, and TNF-α levels were determined by commercial ELISA kits. RESULTS : The mean concentration of IL-1β and TNF-α was significantly higher in macrophage conditioned media of patients with endometriosis (P < 0.02). However, there were no significant changes in peritoneal fluid cytokine levels. Peritoneal macrophage concentrations were also higher in patients with endometriosis. CONCLUSION : This study supports the concept that endometriosis is associated with activation of peritoneal macrophages, and a higher concentration of these cells. This activation is reflected by the increased levels of cytokines found in macrophage conditioned media. The absence of significant changes in peritoneal fluid cytokine levels would seem to indicate that the above derangements are not responsible for the development or progression of endometriosis.     

Social stress enhances IL-1β and TNF-α production by Porphyromonas gingivalis lipopolysaccharide-stimulated CD11b+ cells

Psychological stress is associated with an increased expression of markers of peripheral inflammation, and there is a growing literature describing a link between periodontal pathogens and systemic inflammation. The hypothesis of the present work is that exposing mice to the social stressor, called social disruption (SDR), would enhance the inflammatory response to lipopolysaccharide (LPS) derived from the oral pathogen, Porphyromonas gingivalis. Mice were exposed to SDR for 2 h per day on 6 consecutive days. On the morning following the last cycle of SDR, mice were tested for anxiety-like behavior in the open field test and novel object test. The mice were sacrificed the following day and their spleens harvested. Spleen cells were stimulated with LPS derived from P. gingivalis in the absence or presence of increasing doses of corticosterone. Social disruption resulted in anxiety-like behavior, and the production of IL-1β and TNF-α was significantly higher in spleen cells from mice exposed to SDR in comparison to levels from non-stressed control mice. In addition, the viability of spleen cells from mice exposed to SDR was significantly greater than the viability of cells from non-stressed control mice, even in the presence of high doses of corticosterone. The use of cultures enriched for CD11b+ cells indicated that the stressor was affecting the activity of splenic myeloid cells. This study demonstrates that social stress enhances the inflammatory response to an oral pathogen and could provide a critical clue in the reported associations between stress, inflammation, and oral pathogens.     

Characteristics of IL-6 and TNF-α production by respiratory syncytial virus-infected macrophages in the neonate

The production of IL-6 and TNF-α and the expression of their mRNA were studied with neonatal (cord blood) and adult blood monocyte-derived macrophages (MDM) after in vitro infection with respiratory syncytial virus (RSV). Cord blood MDM exhibited production of high levels of IL-6 within 24 hr after infection. Little or no IL-6 production was detected after 24–48 hr and after in vitro stimulation with inactivated (nonreplicating) virus. Adult blood MDM also produced high levels of IL-6 within 24 hr of RSV infection. Unlike cord blood MDM, adult MDM demonstrated significant activity of IL-6 after 24 hr of infection with live RSV and after exposure to the inactivated virus. The pattern of TNF-α production by cord and adult blood MDM after live RSV infection resembled closely the pattern of IL-6 production. Both cell types produced TNF-α in the first 24 hr after infection. However, little or no production was observed after 24 hr of infection and after exposure to the inactivated virus. The profile of mRNA expression was similar to the production of IL-6 or TNF-α. mRNA expression occurred over a shorter period in cord blood MDM. These observations suggest that inflammatory and immunoregulatory cytokines, such as IL-6 and TNF-α, are produced by neonatal as well as previously primed adult macrophages. However, neonatal cells may be less efficient in inducing IL-6 production. © 1996 Wiley-Liss, Inc.     

Keratinocyte Growth Factor Stimulates Macrophage Inflammatory Protein 3α and Keratinocyte‐derived Chemokine Secretion by Mouse Uterine Epithelial Cells

Citation Haddad SN, Wira CR. Keratinocyte growth factor stimulates macrophage inflammatory protein 3α and keratinocyte‐derived chemokine secretion by mouse uterine epithelial cells. Am J Reprod Immunol 2010; 64: 197–211 Problem Communication between uterine epithelial cells and the underlying stromal fibroblasts is critical for proper endometrial function. Stromal fibroblast‐derived growth factors have been shown to regulate epithelial immune functions. The purpose of this study was to determine whether keratinocyte growth factor (KGF) regulates uterine epithelial cell chemokine and antimicrobial secretion. Method of study Uterine epithelial cells were isolated from Balb/c mice and cultured in either 96‐well plates or transwell inserts. Epithelial cells were treated with KGF, epidermal growth factor (EGF), or hepatocyte growth factor (HGF). Macrophage inflammatory protein 3α (MIP3α) and keratinocyte‐derived chemokine (KC) levels were measured by ELISA. Results Keratinocyte growth factor stimulated the secretion of MIP3α and KC. The effects on MIP3α by KGF were specific because EGF and HGF had no effect. In contrast, KGF, EGF, and HGF had similar effects on KC. Furthermore, KGF administered to the apical side of epithelial cells had no effect on MIP3α or KC secretion, indicating that the KGF receptor is located on the basolateral surface of uterine epithelial cells. Conclusion We demonstrate that KGF plays a role in uterine epithelial cell secretion of MIP3α and KC, key immune mediators involved in the protection of mucosal surfaces in the female reproductive tract.     

Oxidative stress up-regulates IL-8 and TNF-α synthesis by human dendritic cells

In addition to their damaging effects, reactive oxygen intermediates exert a regulatory role on gene expression and cell apoptosis. In this study, we evaluated the effects of oxidative stress on human dendritic cells (DC), a cell type which is critical for the initiation of the immune response. For this purpose, we tested the effects of H₂O₂ on DC derived from adherent peripheral blood mononuclear cells cultured in the presence of granulocyte-macrophage colony-stimulating factor and IL-4. Despite a moderate increase of DC apoptosis in the presence of H₂O₂, we observed that H₂O₂ stimulated the production of IL-8 and TFN-α by DC in a dose-dependent manner. The induction of cytokine synthesis was found to depend on the oxidative properties of H₂O₂ as it was inhibited by the addition of catalase, and to require de novo protein synthesis as it was not observed in the presence of cycloheximide. These data suggest that DC could contribute to innate immunity through an enhanced production of inflammatory cytokines in response to oxidative stress.     

Soluble Receptors Neutralizing TNF-α and IL-1 Block Stress-Triggered Murine Abortion

PROBLEM: In several models of abortion in rodents, the success or failure of the implanted embryos is determined by a balance between pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-2, interleukin-1 (IL-1), and γ-interferon, and cytokines that counteract the former, such as interleukin-10 and transforming growth factor-β2 (TGF-β2)-related suppressor factor. Stress can trigger abortions in susceptible strains of mice and is thought to reflect the pathogenesis of some types of miscarriage in human pregnancy. In mice, stress increases levels of the abortogenic cytokine TNF-α and decreases the suppressive activity of TGF-β2-related factor via a neurotransmitter substance P (SP)-dependent pathway. Evidence for a role of pro-inflammatory cytokines in SP-mediated abortions in vivo is indirect. METHODS: Direct evidence for a role of IL-1 and TNF-α in stress-triggered abortions was sought by injecting pregnant female mice with soluble receptors neutralizing TNF-α (rhuTNFR: Fc) or IL-1 (rmIL-1R) beginning 1 day after implantation and prior to stress. RESULTS: The stress-triggered abortion rate was reduced by 68% when either TNF-α or IL-1 antagonists were injected. The stress-triggered decreased TGF-β2-like suppressive activity in the maternal uterine decidua was not restored by injection of either antagonist; indeed the soluble IL-1 receptor significantly reduced suppressive activity in unstressed control mice, and soluble TNF-α receptor had a lesser effect. CONCLUSIONS: Both IL-1 and TNF-α play a role in the pathogenesis of stress-triggered abortions, and may induce a compensatory physiological increase in suppressive activity in normal pregnancy counteracting pro-inflammatory cytokines.     

Neutrophils fromMycobacterium avium-Infected Mice Produce TNF-α, IL-12, and IL-1β and Have a Putative Role in Early Host Response

Recent evidence supports a role for neutrophils in the host defense againstMycobacterium avium.To determine whether the depletion of neutrophils has an effect on the outcome of infection in mice as determined by the number of bacteria in liver and spleen, we administered RB6-8C5 anti-neutrophil antibody intraperitoneally both early and late in the infection. Mice were then observed for 14 days and harvested. The number of viable bacteria in liver and spleen was determined. While administration of RB6-8C5 antibody early in infection resulted in a significant increase in the number of bacteria in organs when compared with mice receiving immunoglobulin control, administration of RB6-8C5 antibody late in infection (week 3) did not have an impact on the bacterial load in tissue. Infection of CD18 knockout mice (with impaired neutrophil function), however, did not show a significant enhancement ofM. aviumgrowth when compared with that of wild-type control mice. Neutrophils were found to produce increased amounts of TNF-α and IL-12 and IL-1 than control uninfected mice during the initial phase of infection, but not after 2 weeks following infection (although IL-1β levels continue elevated). The results suggest that neutrophils may have a role in the early (innate) immune response againstM. aviumbut it is only evident after acute depletion of neutrophils and not in mice with chronic neutrophil impairment.     

Human γδ T Cells Express a Higher TCR/CD3 Complex Density Than αβ T Cells

The aim of our study was to compare CD3 expression on γδ T cells and αβ T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human γδ T cells constitutively express approximately twofold more of the TCR/CD3 complex than αβ T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of γδ T cells compared to αβ T cells. These clinical laboratory results confirm the fundamental data described elsewhere. γδ T cells deserve further clinical investigations to understand their precise role in human immunity.     

β1C Integrin in Epithelial Cells Correlates with a Nonproliferative Phenotype : Forced Expression of β1C Inhibits Prostate Epithelial Cell Proliferation

The expression of the β_1C integrin, an alternatively spliced variant of the β₁ subunit, was investigated in human adult and fetal tissues. In the adult, β_1C immunoreactivity was found in nonproliferative, differentiated simple, and/or pseudostratified epithelia in prostate glands and liver bile ducts. In contrast, β_1C was undetectable in stratified squamous epithelium of the epidermis and/or in hepatocytes. Luminal prostate epithelial cells expressed β_1C in vivo and in vitro, but no β_1C was seen in basal cells, which are proliferating cells. Fetal prostate expressed β_1C in differentiated glands that had a defined lumen, but not in budding glands, indicating that β_1C is a marker of prostate epithelium differentiation. The β_1C and the common β_1A variants are differentially distributed: β_1A was found in luminal and basal epithelial as well as in stromal cells in the prostate. In the liver, β_1C and β_1A were coexpressed in biliary epithelium, whereas vascular cells expressed only β_1A. Because we found β_1C in nonproliferative and differentiated epithelium, we investigated whether β_1C could have a causal role in inhibiting epithelial cell proliferation. The results showed that exogenous expression of a β_1C, but not of a β_1A, cytoplasmic domain chimeric construct, completely inhibited thymidine incorporation in response to serum by prostate cancer epithelial cells. Consistent with these in vitro results, β_1C appeared to be downregulated in prostate glands that exhibit regenerative features in benign hyperplastic epithelium. These data show that the presence of β_1C integrins in epithelial cells correlates with a nonproliferative, differentiated phenotype and is growth inhibitory to prostate epithelial cells in vitro. These findings indicate a novel pathophysiological role for this integrin variant in epithelial cell proliferation.     

Hydrogen Peroxide Is Crucial for NLRP3 Inflammasome-Mediated IL-1β Production and Cell Death in Pneumococcal Infections of Bronchial Epithelial Cells

Epithelial cells play a crucial role in detection of the pathogens as well as in initiation of the host immune response. Streptococcus pneumoniae (pneumococcus) is a typical colonizer of the human nasopharynx, which can disseminate to the lower respiratory tract and subsequently cause severe invasive diseases such as pneumonia, sepsis, and meningitis. Hydrogen peroxide (H₂O₂) is produced by pneumococci as a product of the pyruvate oxidase SpxB. However, its role as a virulence determinant in pneumococcal infections of the lower respiratory tract is not well understood. In this study, we investigated the role of pneumococcal-derived H₂O₂ in initiating epithelial cell death by analyzing the interplay between 2 key cell death pathways, namely, apoptosis and pyroptosis. We demonstrate that H₂O₂ primes as well as activates the NLRP3 inflammasome and thereby mediates IL-1β production and release. Furthermore, we show that pneumococcal H₂O₂ causes cell death via the activation of both apoptotic as well as pyroptotic pathways which are mediated by the activation of caspase-3/7 and caspase-1, respectively. However, H₂O₂-mediated IL-1β release itself occurs mainly via apoptosis.     

Appearance and distribution of laminin a chain isoforms and integrin α2, α3, α6, β1, and β4 subunits in the developing human small intestinal mucosa

Background: Laminin, a major component of basement membranes, is well known in its classical heterotrimeric form (B1-A-B2) to regulate diverse biological functions, including cell polarization and differentiation. However, the role of merosin, a laminin-like molecule in which an M chain is substituted for its homologous A chain, remains largely unknown. Methods: In the present study, we analyzed by indirect immunofluorescence the expression and distribution of these four laminin chains as well as the integrins α2β1, α3β1,α6β1, and α6β4, four potential recptors, at the epithelial-mesenchymal interface of the developing human small intestine, with a panel of specific monoclonal antibodies. Results: Beginning at 7 weeks of gestation and throughout mucosal organogenesis, the B1 and B2 chains were uniformly detected at the epithelial basement membrane. The A chain also was detected beginning at 7 weeks, and its distribution at the basement membrane remained uniform throughout villus (9+ weeks) and crypt (16+ weeks) formation. In contrast, M chain expression was not observed until 16 weeks; between 16 and 20 weeks, it was exclusively associated with the base of epithelial cells that comprised the forming crypts. Integrins α6β1 and α6β4, as determined by their subunit immunolocalization, appeared to be expressed by all enterocytes from 7 to 20 weeks. In contrast, the expression of the α2β1 and α3β1 integrins was found time- and site-restricted. The α2 subunit was predominantly detected in the epithelial cells of the intervillous area and its derivative, the crypt, whereas the α3 subunit was strongly expressed by all epithelial cells except those located at the bottom of 19–20-week-old crypts. Conclusions: Taken together, these observations demonstrate that both compositional changes in the basement membrane and differential expression of receptors occur during human intestinal organogenesis, suggesting that epithelial cell-matrix interactions play a role during development. © 1995 Wiley-Liss, Inc.     

Gamma-Interferon-Mediated Down-Regulation of Electrolyte Secretion by Intestinal Epithelial Cells: A Local Immune Mechanism?

Active chloride (Cl-) secretion by intestinal crypt enterocytes is the central pathophysiological disturbance in most cases of acute diarrhoea. We examined monolayers of the human intestinal cell line T84 mounted in Ussing chambers to see whether the T-cell lymphokine gamma interferon (IFN-gamma) might affect the Cl- secretory properties of these cells, which morphologically and functionally resemble native crypt enterocytes. Pretreatment of T84 cell layers with IFN-gamma for 24 h (but not for 3 h) markedly decreased the Cl- secretory response to vaso-active intestinal polypeptide (VIP) and to cholera toxin and carbachol without appreciably affecting the overall morphology, electrical resistance, or cyclic AMP response of the T84 cell monolayer. The IFN-gamma treatment, however, did induce subtle changes in the T84 cell membrane protein composition which might have affected ion channels regulating Cl- secretion. Our results may indicate a possible novel 'cell-mediated' immune mechanism through which activated gut T cells could modulate the extent of intestinal electrolyte and fluid secretion in, for example, enteric infections.     

Metabolic Actions in Intestinal Smooth Muscle Associated with Relaxation Mediated by Adrenergic α- and β-Receptors

In colonic muscle from the rabbit a relaxing effect was induced by stimulation both of adrenergic α-receptors with phenylephrine and of β-receptors with isoprenaline. The α-receptor induced relaxation was not accompanied initially by any metabolic effects. After a latency period of 3 min did a decrease in the phosphorylase α activity, a decrease in the concentrations of hexose phosphates and lactate and an increase in the concentration of high-energy phosphate compounds (ATP and CrP) become evident. There was a decrease of the cyclic AMP content. These effects were inhibited by dibenamine. The relaxation induced via adrenergic β-receptors was preceded by an increase in the cyclic AMP content and the phosphorylase α activity, an increase in the concentration of hexose phosphates and lactate and an initial decrease of the ATP and CrP concentrations, which after 10 min was succeeded by an increase. The magnitudes of the relaxing and metabolic responses following stimulation of preceptors were correlated with regard to time and dose. These responses were blocked by an adrenergic β-receptor blocking agent (sotalol) and were also eliminated in carbohydrate-poor muscle. The relaxing and metabolic effects of isoprenaline could be reproduced completely by cyclic AMP, only incompletely by 5'AMP and not at all by other tested cyclic nucleotides.