Heme oxygenase-1 is a critical regulator of nitric oxide production in enterohemorrhagic Escherichia coli-infected human enterocytes

Enterohemorrhagic Escherichia coli (EHEC) are the causative agent of hemolytic-uremic syndrome. In the first stage of the infection, EHEC interact with human enterocytes to modulate the innate immune response. Inducible NO synthase (iNOS)-derived NO is a critical mediator of the inflammatory response of the infected intestinal mucosa. We therefore aimed to analyze the role of EHEC on iNOS induction in human epithelial cell lines. In this regard, we show that EHEC down-regulate IFN-gamma-induced iNOS mRNA expression and NO production in Hct-8, Caco-2, and T84 cells. This inhibitory effect occurs through the decrease of STAT-1 activation. In parallel, we demonstrate that EHEC stimulate the rapid inducible expression of the gene hmox-1 that encodes for the enzyme heme oxygenase-1 (HO-1). Knock-down of hmox-1 gene expression by small interfering RNA or the blockade of HO-1 activity by zinc protoporphyrin IX abrogated the EHEC-dependent inhibition of STAT-1 activation and iNOS mRNA expression in activated human enterocytes. These results highlight a new strategy elaborated by EHEC to control the host innate immune response.

Pregnancy induces numerical and functional changes of CD4+CD25 high regulatory T cells in patients with rheumatoid arthritis

OBJECTIVE: In a prospective study we investigated whether numerical and functional changes of CD4+CD25(high) regulatory T cells (Treg) were associated with changes of disease activity observed during pregnancy and post partum in patients with rheumatoid arthritis (RA). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 12 patients with RA and 14 healthy women during and after pregnancy. Fluorescence-activated cell sorting (FACS) was used to sort CD4+CD25(high) T cells and CD4+CD25- T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies alone or in co-culture to investigate proliferation and cytokine secretion. RESULTS: Frequencies of CD4+CD25(high) Treg were significantly higher in the third trimester compared to 8 weeks post partum in patients and controls. Numbers of CD4+CD25(high) Treg inversely correlated with disease activity in the third trimester and post partum. In co-culture experiments significantly higher amounts of IL10 and lowered levels of tumour necrosis factor (TNF)alpha and interferon (IFN)gamma were found in supernatants of the third trimester compared to postpartum samples. These findings were independent from health or disease in pregnancy, however postpartum TNFalpha and IFN gamma levels were higher in patients with disease flares. CONCLUSION: The amelioration of disease activity in the third trimester corresponded to the increased number of Treg that induced a pronounced anti-inflammatory cytokine milieu. The pregnancy related quantitative and qualitative changes of Treg suggest a beneficial effect of Treg on disease activity.

Clinical and immunologic effects of H1 antihistamine preventive medication during honeybee venom immunotherapy

BACKGROUND: H1 antihistamines increase safety during allergen-specific immunotherapy and might influence the outcome because of immunoregulatory effects. OBJECTIVE: We sought to analyze the influence of 5 mg of levocetirizine (LC) on the safety, efficacy, and immunologic effects of ultrarush honeybee venom immunotherapy (BVIT). METHOD: In a double-blind, placebo-controlled study 54 patients with honeybee venom allergy received LC or placebo from 2 days before BVIT to day 21. Side effects during dose increase and systemic allergic reactions (SARs) to a sting challenge after 120 days were analyzed. Allergen-specific immune response was investigated in skin, serum, and allergen-stimulated T-cell cultures. RESULTS: Side effects were significantly more frequent in patients receiving placebo. Four patients receiving placebo dropped out because of side effects. SARs to the sting challenge occurred in 8 patients (6 in the LC group and 2 in the placebo group). Seven SARs were only cutaneous, and 1 in the placebo group was also respiratory. Difference of SARs caused by the sting challenge was insignificant. Specific IgG levels increased significantly in both groups. Major allergen phospholipase A(2)-stimulated T cells from both groups showed a slightly decreased proliferation. The decrease in IFN-gamma and IL-13 levels with placebo was not prominent with LC, whereas IL-10 levels showed a significant increase in the LC group only. Decreased histamine receptor (HR)1/HR2 ratio in allergen-specific T cells on day 21 in the placebo group was prevented by LC. CONCLUSIONS: LC reduces side effects during dose increase without influencing the efficacy of BVIT. LC modulates the natural course of allergen-specific immune response and affects the expression of HRs and cytokine production by allergen-specific T cells.

Evaluating the potential of IP-10 and MCP-2 as biomarkers for the diagnosis of tuberculosis

The aim of the present study was to evaluate the potential of diagnostic tests based on interferon-gamma inducible protein (IP)-10 and monocyte chemotactic protein (MCP)-2, and compare the performance with the QuantiFERON TB Gold In-Tube (QFT-IT; Cellestis, Carnagie, Australia) test. IP-10 and MCP-2 were determined in supernatants from whole blood stimulated with Mycobacterium tuberculosis-specific antigens. Samples were obtained from 80 patients with culture- and/or PCR-proven tuberculosis (TB), and 124 unexposed healthy controls: 86 high school students and 38 high school staff. IP-10 and MCP-2 test cut-offs were established based on receiver operating characteristic curve analysis. TB patients produced significantly higher levels (median) of IP-10 (2158 pg x mL(-1)) and MCP-2 (379 pg x mL(-1)) compared with interferon (IFN)-gamma (215 pg x mL(-1)). The QFT-IT, IP-10 and MCP-2 tests detected 81, 83 and 71% of the TB patients; 0, 3 and 0% of the high school students and 0, 16 and 3% of the staff, respectively. Agreement between tests was high (>89%). By combining IP-10 and IFN-gamma tests, the detection rate increased among TB patients to 90% without a significant increase in positive responders among the students. In conclusion, interferon-gamma inducible protein-10 and monocyte chemotactic protein-2 responses to Mycobacterium tuberculosis-specific antigens could be used to diagnose infection. Combining interferon-gamma inducible protein-10 and interferon-gamma may be a simple approach to increase the detection rate of the Mycobacterium tuberculosis-specific in vitro tests.

Host responses to intestinal microbial antigens in gluten-sensitive mice

BACKGROUND AND AIMS: Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice. METHODOLOGY/PRINCIPAL FINDINGS: HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-gamma in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota. CONCLUSION: Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-gamma production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.

Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites

We investigated the protective potential of recombinant his-tagged antigens recNcMIC1, recNcMIC3 and recNcROP2, applied either as single vaccines or as vaccine combinations, in BALB/c mouse models for cerebral and fetal infection. Subsequently, mice were mated and challenged by i.p. inoculation of 2 x 10(6)Neospora caninum tachyzoites at day 7 of pregnancy. The mortality and morbidity of adult mice (non-pregnant and dams) and of the newborn pups was studied for a period of 40 days following birth. Vaccination of non-pregnant mice with recNcROP2 or combinations of recNcROP2 with recNcMIC antigens significantly reduced the numbers of mice suffering from clinical signs, and morbidity was completely prevented with the combination of all three antigens. Of the dams, the groups receiving either recNcROP2 alone or the combination of all three antigens did not exhibit any morbidity, the groups receiving ROP2 mixed with either MIC1 or MIC3 exhibited reduced numbers of deaths, and in the infection control group and the adjuvant group 50% and 43% of mice, respectively, succumbed to disease. For pups, the highest survival rates were noted for the groups receiving recNcROP2 (50%) and recNcROP2/NcMIC1/NcMIC3 (35%), while in the infection- and adjuvant- control groups all pups died, the latest at days 25 and 30, respectively. Quantification of parasite DNA by N. caninum-specific real-time PCR revealed consistently lower parasite burdens in brain tissue of pups from vaccinated groups compared with the controls. However, dense granule antigen 2 (GRA2) real-time reverse transcriptase-PCR on brain tissue of surviving pups (applied here to detect viable parasites) demonstrated that only the pups from the group vaccinated with all three antigens in combination appeared free of viable tachyzoites, while in all other groups viable parasites were still present. Serological analysis of humoral (total IgG, IgG1 and IgG2a) and serum cytokine (IL-4 and IFN-gamma) responses showed that this effect was associated with a Th-2-biased immune response, with a clearly elevated IL-4/IFN-gamma ratio in the mice receiving all three antigens in combination. In conclusion, a mixture of recombinant antigens representing important secretory micronemal and rhoptry proteins leads to a significant protection against vertical transmission of N. caninum in mice.

Human bronchial epithelium controls TH2 responses by TH1-induced, nitric oxide-mediated STAT5 dephosphorylation: implications for the pathogenesis of asthma

Increased levels of NO in exhaled air in association with increased NO synthetase (NOS)2 expression in bronchial epithelial are hallmark features of asthma. It has been suggested that NO contributes to asthma pathogenesis by selective down-regulation of TH1 responses. We demonstrate, however, that NO can reversibly limit in vitro expansion of both human TH1 and TH2 CD4+ T cells. Mechanistically, NO induces cGMP-mediated reversible STAT5 dephosphorylation and therefore interferes with the IL-2R activation cascade. Human bronchial epithelial cells (HBEC) up-regulate NOS2 after stimulation with IFN-gamma secreted by TH1 CD4+ T cells and release NO, which inhibits both TH1 and TH2 cell proliferation. This reversible T cell growth arrest depends on NO because T cell proliferation is completely restored after in vitro blocking of NOS2 on HBEC. HBEC thus drive the effector end of a TH1-controlled feedback loop, which protects airway mucosal tissues at the potential lesional site in asthma from overwhelming CD4+ TH2 (and potentially TH1) responses following allergen exposure. Variations in the efficiency of this feedback loop provides a plausible mechanism to explain why only a subset of atopics sensitized to ubiquitous aeroallergens progress to expression of clinically relevant levels of airways inflammation.

Depressed type 1 cytokine synthesis by superantigen-activated CD4+ T cells of women with human papillomavirus-related high-grade squamous intraepithelial lesions.

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4(+) T and CD8(+) T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4(+) T cells that produced IL-2 (P = 0.045), IFN-gamma (P = 0.040), and TNF-alpha (P = 0.015) and a significantly lower percentage of activated CD8(+) T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4(+) T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8(+) T cells.

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24.

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.

Differentiative requirements for human CD8$\sp+$ suppressor lymphocytes

The aim of this research was to characterize the differentiative requirements of human CD8$\sp{+}$ suppressor lymphocytes. We investigated the role of monocytes in cellular interactions required for generation of T suppressor cells (Ts) in pokeweed mitogen (PWM) stimulated peripheral blood mononuclear cells (PBMC). We observed that the functional activity of CD8$\sp{+}$ T cells was dependent on the concentration of monocytes in the inductive cultures; at concentrations normally present in peripheral blood, PWM stimulation induced potent suppressor activity, whereas under conditions of moderate monocyte depletion the same phenotypic subset of CD8$\sp{+}$ cells enhanced responses. We also demonstrated that differentiation of CD8$\sp{+}$CD28$\sp{-}$ suppressor cells could be mediated by soluble products elaborated by monocytes and CD4$\sp{+}$ cells, identified as PGE$\sb2$ and IFN$\gamma$ respectively. These two signals were required sequentially to cause Ts induction. That is PGE$\sb2$ was required initially, followed by an IFN$\gamma$-dependent differentiative step. We also explored the possibility that PGE$\sb2$ caused modulation of the IFN$\gamma$ receptor number and/or affinity on CD8$\sp{+}$ cells, which might render these cells responsive to the differentiative effect of the IFN$\gamma$-signal. Using radiolabelled $\sp{125}$I-IFN$\gamma$, direct binding assays demonstrated that 10$\sp{-8}$M PGE$\sb2$ selectively increased the number of receptors on the CD8$\sp{+}$ cells. In contrast CD4$\sp{+}$ cells treated similarly exhibited no significant change in their number of IFN$\gamma$ receptors. These results, thus, suggest a relationship between PGE$\sb2$ induced expression of IFN$\gamma$ receptor and the initial requirement for PGE$\sb2$ in IFN$\gamma$-dependent differentiation of Ts cells. Together, our results suggest a crucial role for PGE$\sb2$ and IFN$\gamma$ in regulation of the immune response. Furthermore, such detailed definition of the differentiative requirements for CD8$\sp{+}$ suppressor cells should provide new insight into fundamental mechanisms of immunoregulation. ^

Transcriptional regulation of the invariant chain associated with MHC class II molecules

The invariant chain associated with the major histocompatibility complex (MHC) class II molecules is a non-polymorphic glycoprotein implicated in antigen processing and class II molecule intracellular transport. Class II molecules and invariant chain (In) are expressed primarily by B lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon gamma (IFN-$\gamma$) in a variety of cell types such as endothelial cells, fibroblasts, and astrocytes. In this study the cis-acting sequences involved in the constitutive, tissue-specific, and IFN-$\gamma$ induced expression of the human In gene were investigated and nuclear proteins which specifically bound these sequences were identified.^ To define promoter sequences involved in the regulation of the human In gene, 790 bp 5$\sp\prime$ to the initiation of transcription were subcloned upstream of the gene encoding chloramphenicol acetyl transferase (CAT). Transfection of this construct into In expressing and non-expressing cell lines demonstrated that this 790 bp In promoter sequence conferred tissue specificity to the CAT gene. Deletion mutants were created in the promoter to identify sequences important for transcription. Three regulatory regions were identified $-$396 to $-$241, $-$241 to $-$216, and $-$216 to $-$165 bp 5$\sp\prime$ to the cap site. Transfection into a human glioblastoma cell line, U-373 MG, and treatment with IFN-$\gamma$, demonstrated that this 5$\sp\prime$ region is responsive to IFN-$\gamma$. An IFN-$\gamma$ response element was sublocalized to the region $-$120 to $-$61 bp. This region contains homology to the interferon-stimulated response element (ISRE) identified in other IFN responsive genes. IFN-$\gamma$ induces a sequence-specific DNA binding factor which binds to an oligonucleotide corresponding to $-$107 to $-$79 bp of the In promoter. This factor also binds to an oligonucleotide corresponding to $-$91 to $-$62 of the interferon-$\beta$ gene promoter, suggesting this factor may be member of the IRF-1/ISGF2, IRF-2, ICSBP family of ISRE binding proteins. A transcriptional enhancer was identified in the first intron of the In gene. This element, located in a 2.6 kb BamHI/PstI fragment, enhances the IFN-$\gamma$ response of the promoter in U-373 MG. The majority of the In enhancer activity was sublocalized to a 550 bp region $\sim$1.6 kb downstream of the In transcriptional start site. ^

Host factors in metastasis: Direct lysis of bloodborne metastatic tumor cells by murine vascular endothelial cells

During the process of cancer metastasis, the majority of circulating tumor cells arrest in microcapillary beds and then rapidly die. To study whether vascular endothelial cells can directly lyse tumor cells, we isolated vascular endothelial cells by perfusion of lungs from immunocompetent or nude mice. The cells were grown in culture, and then cloned and characterized. Cloned endothelial cells were incubated with several lymphokines and cytokines. Cells incubated with IFN-$\gamma$ and TNF lysed a variety of tumor cells with different metastatic potential. Mouse skin and lung fibroblasts treated with the same cytokines did not. Endothelial cell mediated tumor cell lysis was not due to different binding ability of tumor cells to cytokine treated and untreated endothelial monolayers. Kinetic studies demonstrated that the continuous presence of cytokines in the tumor-endothelial cocultures was necessary to produce maximal lysis of tumor cells. Target cell lysis was not due to the direct effects of IFN-$\gamma$ or TNF, since vascular endothelial cells isolated from the lung of nude mice lysed human melanoma cells that are sensitive or resistant to TNF. Cytokine treated endothelial cells produced a high level of nitric oxide, which is known to be cytotoxic to a variety of target cells. The level of nitric oxide production was directly correlated with the degree of tumor cell lysis. A specific inhibitor of nitric oxide synthesis(N$\sp{\rm G}$-monomethyl-L-arginine), completely inhibited production of nitric oxide and tumor cell lysis. Treatment of cytokine activated endothelial cells with dexamethasone also inhibited tumor cell lysis. This inhibition was independent of tumor-endothelial adhesion but correlated with inhibition of nitric oxide production. Collectively, these results suggest that vascular endothelial cells can directly destory tumor emboli and thus play an active role in the pathogenesis of cancer metastasis. ^

The role of nitric oxide in apoptosis and cancer metastasis

The purpose of these studies was to investigate the role of nitric oxide (NO) in tumor metastasis. K-1735 Metastatic cells survived in blood circulation to produce experimental lung metastases, whereas nonmetastatic cells did not. After incubation with combination cytokines or lipopolysaccharide (LPS), nonmetastatic cells exhibited high levels of inducible nitric oxide synthase (iNOS) activity and NO production, whereas metastatic cells did not. The production of NO directly correlated with cytotoxic effects of cytokines or LPS. To provide direct evidence for the inverse correlation between the production of endogenous NO and the ability of K-1735 cells to survive in syngeneic mice to produce lung metastases, highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containing 3 mM NMA. C4.P, C4.Neo.3, and C4.S2.3 cells were highly metastatic whereas C4.L8.5 cells were not metastatic. The C4.L8.5 cells produced slow growing subcutaneous tumors in nude mice, whereas the other three lines produced fast growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells induced apoptosis. Collectively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis.^ Furthermore, multiple systemic administrations of multilamellar vesicle-liposomes (MLV) containing the lipopeptide CGP 31362 (MLV-31362) or MLV-31362 combined with murine interferon-gamma (IFN-$\gamma$) eradicated the metastases by M5076 reticular cell sarcoma. Tumor regression correlated with iNOS expression within the tumor lesions and with increased NO production. The administration of NMA significantly decreased NO production and diminished the antitumor activities. These data imply that the activation of iNOS can serve as a target for immunotherapeutic agents for treatment of murine reticulum cell sarcoma metastases. ^

Expression and regulation of interleukins in human head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate abnormal cell-mediated immunity which is most pronounced at the primary tumor site. Therefore, we tested whether this aberrant immunity could be due to tumor-derived cytokines. We investigated the presence of cytokine mRNA and protein in 8 HNSCC-derived cell lines; RT-PCR results indicated mRNA's for IL-1$\alpha$ and TGF-$\alpha$ (8/8), TGF-$\beta$ (7/8), IL-1$\beta$ (7/8), IL-4 and IL-6 (4/8). IL-2, IFN-$\gamma,$ and TNF-$\alpha$ mRNA was not detected. Supernatants from 6 of these cell lines were analyzed by ELISA and IL-1$\alpha,$ IL-1$\beta,$ and IL-6 were markedly increased compared to HPV-16 immortalized human oral keratinocytes. IL-1$\alpha$ was found in the highest concentration $>$IL-6 $>$ IL-1$\beta.$^ To approach the mechanisms of cytokine regulation, 4 cell lines were compared for HPV DNA presence, p53 status, and cytokine expression. An association between HPV DNA and cytokine expression was not found. However, cell lines secreting the most IL-6 had mutant p53 and/or HPV 16 E6/E7 expression. Further regulatory investigations revealed that exogenous IL-1$\alpha$ and/or IL-1$\beta$ minimally stimulated the proliferation of 2/3 cell lines, as well as strongly induced IL-6 production in 3/3; this effect was completely abrogated by IL-1Ra. IL-1Ra also inhibited the secretion of IL-1$\alpha$ and IL-1$\beta$ in 2/3 cell lines. These data suggest an IL-1 autocrine loop in certain HNSCC cell lines. Because IL-2 induces IL-1 and is used in therapy of HNSCC, the expression of IL-2 receptor was also investigated; IL-2 $\alpha$ and $\beta$ subunits were detected in 3/3 cell lines and $\gamma$ subunits was detected in one. Exogenous IL-2 inhibited the proliferation, but stimulated the secretion of IL-1$\alpha$ in 2/3, and IL-1$\beta$ and IL-6 in 1/3 cell lines.^ To determine if our cell line findings were applicable to patients, immunohistochemistry was performed on biopsies from 12 invasive tumors. Unexpectedly, universal intracellular production of IL-1$\alpha,$ IL-1$\beta,$ and IL-6 protein was detected. Therefore, the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients. ^

Suppression of T lymphocyte activation in simulated microgravity

Immune dysfunction is encountered during spaceflight. Various aspects of spaceflight, including microgravity, cosmic radiation, and both physiological and psychological stress, may perturb immune function. We sought to understand the impact of microgravity alone on the cellular mechanisms critical to immunity. Clinostatic RWV bioreactors that simulate aspects of microgravity were used to analyze the response of human PBMC to polyclonal and oligoclonal activation. PHA responsiveness in the RWV bioreactor was almost completely diminished. IL-2 and IFN-$\gamma$ secretion was reduced whereas IL-1$\beta$ and IL-6 secretion was increased, suggesting that monocytes may not be as adversely affected by simulated microgravity as T cells. Activation marker expression (CD25, CD69, CD71) was significantly reduced in RWV cultures. Furthermore, addition of exogenous IL-2 to these cultures did not restore proliferation. Antigen specific T cell activation, including the mixed-lymphocyte reaction, tetanus toxoid responsiveness, and Borrelia activation of a specific T cell line, was also suppressed in the RWV bioreactor.^ The role of altered culture conditions in the suppression of T cell activation were considered. Potential reduced cell-cell and cell-substratum interactions in the RWV bioreactor may play a role in the loss of PHA responsiveness. However, PHA activation in Teflon culture bags that limit cell-substratum interactions was not affected. Furthermore, increasing cell-population density, and therefore cell-cell interactions, in the RWV cultures did not help restore PHA activation. However, placing PBMC within small collagen beads did partially restore PHA responsiveness. Finally, activation of purified T cells with crosslinked CD2/CD28 or CD3/CD28 antibody pairs, which does not require costimulation through cell-cell contact, was completely suppressed in the RWV bioreactor suggesting a defect internal to the T cell.^ Activation of both PBMC and purified T cells with PMA and ionomycin was unaffected by RWV culture, indicating that signaling mechanisms downstream of PKC activation and calcium flux are not sensitive to simulated microgravity. Furthermore, sub-mitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus, our data indicate that during polyclonal activation in simulated microgravity, there is a specific dysfunction within the T cell involving the signaling pathways upstream of PKC activation. ^