Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot.

Background: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis on whether the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. Methods: Mice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. Results: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted. Conclusion: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

Antidiabetogenic effect of pentoxifylline is associated with systemic and target tissue modulation of cytokines and nitric oxide production.

We have shown recently that xanthine derivative pentoxifylline (PTX) downregulates an inflammatory autoimmune process triggered in genetically susceptible Dark Agouti rats by multiple low doses of streptozotocin (MLD-SZ, 20 mg/kg/day ip for 5 days). We studied the cellular and molecular consequences of PTX treatment during MLD-SZ-induced diabetes with special emphasis on local vs. systemic production of inflammatory mediators. Administration of PTX (200 mg/kg/day for 10 days) during induction of the disease reduced clinical signs of diabetes and protected rats from development of destructive intrainsulitis. Pentoxifylline did not affect diabetogenic effect of single high dose of SZ (100 mg/kg SZ). Ex vivo analysis of the islets of Langerhans performed in early disease development revealed that PTX downregulates production of proinflammatory cytokines IFN-gamma and TNF, as well as inducible nitric oxide synthase (iNOS) expression and NO production. In addition, PTX treatment suppressed splenocyte autoreactivity, as well as the frequency of cells expressing IL-2R and MHC class II antigens. There was no evidence of any changes in proportion of ICAM-1 and LFA-1 expressing splenocytes in comparison to control MLD-SZ-treated animals. In contrast to suppressed intraislet production, high peripheral expression of both iNOS mRNA and NO was found in MLD-SZ rats treated with PTX. Taken together, the data indicate that the effect on both systemic and intra-islet production of NO, suppression of autoreactive cell activation and of local type 1 cytokine release may contribute to the therapeutic benefit achieved by PTX in the rat.

Detection and analysis of antigen-specific CD8+ T cells.

Based on recent advances in techniques that can detect and enumerate antigen-specific CD8+ T cells, it is evident that these cells can differentially regulate CD8+ T cell effector mechanisms at the single-cell level. Interplay between effector mechanisms that are employed by antigen-specific CD8+ T cells during the immune response in vivo can be addressed with different techniques that "count" cells either directly (T cell receptor (TCR) expression) or indirectly (antigen-specific cytokine production).

Cutting edge: OFF cycling of TNF production by antigen-specific CD8+ T cells is antigen independent.

Although they are known for their capacity to kill infected cells, Ag-specific CD8(+) T cells elaborate other effector mechanisms, including TNF and IFN-gamma, that contribute to defense against infection. Ag-specific CD8(+) T cells rapidly turn ON and turn OFF IFN-gamma production in direct response to Ag contact, presumably to minimize the potential immunopathology that could result from inappropriate secretion of this inflammatory mediator. In this study, we show, using in vitro propagated and directly ex vivo-analyzed Ag-specific CD8(+) T cells, that in contrast to Ag-dependent ON/OFF cycling of IFN-gamma production, the cessation of TNF production by the same IFN-gamma producing cells is rapid and Ag independent.

Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferon-gamma.

T cell memory depends on factors that regulate expansion and death of these cells after antigenic stimulation. Mice deficient in perforin and interferon-gamma (IFN-gamma) exhibited increased expansion, altered immunodominance, and decreased death of antigen-specific CD8+ T cells after infection with an attenuated strain of Listeria monocytogenes, which was cleared from these mice. Expansion of CD8+ T cells was controlled by perforin, whereas IFN-gamma regulated immunodominance and the death phase. Thus, perforin and IFN-gamma regulate distinct elements of CD8+ T cell homeostasis independently of their role as antimicrobial effector molecules.

Adaptive immunity against Listeria monocytogenes in the absence of type I tumor necrosis factor receptor p55.

Tumor necrosis factor (TNF) and the type I TNF receptor (TNFRI), p55, are critical for resistance against primary infections with the intracellular bacterial pathogen Listeria monocytogenes. Importantly, however, susceptibility to primary listeriosis in cytokine-deficient mice does not preclude the development or expression of effective adaptive immunity against virulent L. monocytogenes. We used TNFRI(-/-) mice to study adaptive antilisterial immunity in the absence of interactions between TNF and TNFRI. Our experiments indicate that TNFRI(-/-) mice survive and clear high-dose challenges with an attenuated strain of L. monocytogenes that is incapable of cell-to-cell spread. Furthermore, TNFRI(-/-) mice immunized with attenuated L. monocytogenes go on to develop potent adaptive immunity to subsequent high-dose challenges with virulent L. monocytogenes. Interestingly, CD8(+) T-cell depletion in vivo inhibits immunity to L. monocytogenes in the spleen but not in the liver of TNFRI(-/-) mice. The adaptive immune response in these animals is characterized by activation of listeriolysin O-specific CD8(+) T cells, which are capable of transferring antilisterial immunity to naive wild-type C57BL/6 host mice. These experiments demonstrate the development and expression of potent CD8(+) T-cell-mediated antilisterial immunity in the absence of TNFRI.

Nitric oxide promotes growth and major histocompatibility complex-unrestricted cytotoxicity of interleukin-2-activated rat lymphocytes.

The effect of nitric oxide (NO) on growth and major histocompatibility complex(MHC)-unrestricted cytotoxicity of interleukin(IL)-2-cultivated rat spleen nonadherent mononuclear cells was examined. NO donor sodium nitroprusside (SNP) at relatively low concentrations increased magnitude, as well as duration of IL-2-induced proliferative response of nonadherent splenocytes. SNP effect depended completely on released NO, because it was prevented by NO scavenger haemoglobin, but not mimicked by expired SNP solution, unable to generate NO, or ferricyanide, a second breakdown product of SNP. Other NO donors - SIN-1, SNAP and GSNO failed to exert SNP-like growth-enhancing action, probably as a consequence of rapid NO generation, compared to sustained NO release by SNP. All NO-releasing chemicals at high concentration blocked IL-2-induced proliferation. Growth-promoting effect of SNP-derived NO was independent of guanilat cyclase activation, because dibutyryl cGMP did not affect IL-2-triggered splenocyte proliferation. Macrophage NO acted in a manner similar to SNP; at low concentrations it promoted IL-2-induced splenocyte growth, however higher amounts were suppressive. Cytotoxicity of IL-2-activated splenocytes against NK-sensitive K562 cell line was significantly increased when SNP was present during cultivation with IL-2. Proportion of NKR-P1+ and CD25+ cells, as well as per cell expression of these important activation molecules were increased upon SNP treatment, suggesting possible mechanism for the observed NO action.

Adaptive immunity and enhanced CD8+ T cell response to Listeria monocytogenes in the absence of perforin and IFN-gamma.

Single Ag-specific CD8+ T cells from IFN-gamma-deficient (GKO) or perforin-deficient (PKO) mice provide substantial immunity against murine infection with Listeria monocytogenes. To address the potential for redundancy between perforin and IFN-gamma as CD8+ T cell effector mechanisms, we generated perforin/IFN-gamma (PKO/GKO) double-deficient mice. PKO/GKO-derived CD8+ T cells specific for the immunodominant listeriolysin O (LLO91-99) epitope provide immunity to LM infection similar to that provided by Ag-matched wild-type (WT) CD8+ T cells in the liver but reduced in the spleen. Strikingly, polyclonal CD8+ T cells from immunized PKO/GKO mice were approximately 100-fold more potent in reducing bacterial numbers than the same number of polyclonal CD8+ T cells from immunized WT mice. This result is probably quantitative, because the frequency of the CD8+ T cell response against the immunodominant LLO91-99 epitope is >4.5-fold higher in PKO/GKO mice than WT mice at 7 days after identical immunizations. Moreover, PKO/GKO mice can be immunized by a single infection with attenuated Listeria to resist >80,000-fold higher challenges with virulent organisms than naive PKO/GKO mice. These data demonstrate that neither perforin nor IFN-gamma is required for the development or expression of adaptive immunity to LM. In addition, the results suggest the potential for perforin and IFN-gamma to regulate the magnitude of the CD8+ T cell response to infection.

Cutting edge: antilisterial activity of CD8+ T cells derived from TNF-deficient and TNF/perforin double-deficient mice.

The mechanisms by which CD8+ T cells mediate immunity against bacterial pathogens remain largely unknown. Perforin-dependent cytolysis plays a role, but is not required for CD8+ T cell-mediated immunity against Listeria monocytogenes. TNF is essential for CD8+ T cell immunity to L. monocytogenes, but the cellular source of TNF is undefined. TNF-deficient and TNF/perforin double-deficient mice were used to generate CD8+ T cells specific for an L. monocytogenes-derived Ag. Wild-type and TNF-deficient CD8+ T cells mediated antilisterial immunity in wild-type but not TNF-deficient host mice, revealing that CD8+ T cell-derived TNF is not required for CD8+ T cell-mediated antilisterial immunity, but demonstrating a role for TNF derived from other cell types. TNF/perforin double-deficient CD8+ T cells mediated antilisterial immunity in the liver, but not in the spleen, of wild-type recipient mice, suggesting that perforin-independent immunity in the spleen requires CD8+ T cell-derived TNF.

Intracellular staining for TNF and IFN-gamma detects different frequencies of antigen-specific CD8(+) T cells.

CD8(+) T lymphocytes are important mediators of adaptive immunity against certain viral, protozoan and bacterial pathogens. Activated CD8(+) T cells are able to induce cytolysis of infected cells (perforin and CD95-CD95L mediated pathways) and also elaborate cytokines, including IFN-gamma and TNF after appropriate MHC class I-peptide recognition. New technologies for the detection of antigen-specific CD8(+) T cells, including tetrameric MHC class I-peptide complexes, intracellular IFN-gamma staining and IFN-gamma ELISPOT analysis have revised our understanding of the magnitude of the CD8(+) T cell response to infection. Here, using intracellular cytokine staining, we compare detection of IFN-gamma and TNF in the analysis of pathogen-specific CD8(+) T cell lines and CD8(+) T cells after primary viral infection (LCMV) or secondary bacterial infection (Listeria monocytogenes). Under multiple conditions and with multiple epitopes, we find that staining for intracellular IFN-gamma consistently detects a higher frequency of antigen-specific CD8(+) T cells than detection of intracellular TNF. However, (a) intracellular staining for TNF can be used to detect antigen-specific CD8(+) T cell responses and (b) intracellular staining for cytokines is a useful approach for in vitro characterization of antigen-specific CD8(+) T cell lines.

Cryptococcus neoformans neutralizes macrophage and astrocyte derived nitric oxide without interfering with inducible nitric oxide synthase induction or catalytic activity - possible involvement of nitric oxide consumption.

The effect of Cryptococcus neoformans on the accumulation of nitrite, an indicator of nitric oxide (NO) synthesis, was investigated in cytokine (interferon-gamma [IFN-gamma] and interleukin [IL]-1)-stimulated cultures of rat peritoneal macrophages and C6 astrocytoma cells. Cytokine-induced nitrite generation in cultures of both cell types was inhibited in a dose-dependent manner by live C. neoformans, but not by heat-killed cryptococcal cells or conditioned medium from yeast cultures. C. neoformans-mediated reduction of nitrite formation coincided with impairment of NO-dependent macrophage tumoricidal activity. Cytokine-triggered induction of inducible NO synthase (iNOS) was unaffected in C6 cells, and only marginally reduced in macrophages. When cells were pretreated with cytokines for 24 h to induce iNOS, and any further induction was prevented by inhibition of protein synthesis, C. neoformans was still able to reduce nitrite accumulation in cultures of both cell types. Finally, live C. neoformans, but not heat-killed yeast cells or yeast culture supernatant, significantly reduced nitrite production in a culture solution of NO-releasing compound S-nitrosoglutathione (GSNO). Thus, it appears that cryptococcal reduction of nitrite formation in macrophage and C6 cultures was caused by the consumption of NO by some yeast molecule, rather than by the inhibition of cellular NO synthesis.

Cyclosporin A suppresses the induction of nitric oxide synthesis in interferon-gamma-treated L929 fibroblasts.

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) activity in murine L929 fibroblasts were investigated. IFN-gamma-induced NO production in L929 cells was mediated through an iNOS-dependent L-arginine-NO pathway, since it was abrogated by a selective inhibitor of iNOS, aminoguanidine. CsA applied simultaneously with IFN-gamma caused a dose-dependent reduction of NO synthesis in L929 cells. However, CsA did not influence the enzymatic activity of iNOS, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-gamma and any further induction was blocked by the protein-synthesis inhibitor cycloheximide. IFN-gamma-triggered expression of mRNA for interferon regulatory factor-1 was not reduced by CsA-treatment, suggesting that this iNOS transcription factor is not a target in CsA-mediated inhibition of NO synthesis. Finally, FK506 was not able to mimic the inhibitory effect of CsA on NO production in L929 cells, indicating the calcineurin-independent mechanism of CsA action. These results indicate that CsA suppresses NO synthesis in L929 cells independent of calcineurin inhibition, and interfering with intracellular pathways involved in the iNOS induction, rather than inhibiting its enzymatic activity.

Cyclosporin A inhibits activation of inducible nitric oxide synthase in C6 glioma cell line.

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in rat C6 glioma cell line were investigated. CsA applied simultaneously with iNOS activator IFN-gamma caused dose-dependent reduction of NO synthesis in confluent C6 cells, as determined by measuring accumulation of nitrite, an indicator of NO production, in 48 h culture supernatants. IFN-gamma-induced expression of iNOS, but not interferon regulatory factor-1 (IRF-1) mRNA was reduced in CsA-treated cells. The enzymatic activity of iNOS was not changed by CsA, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-gamma and any further induction was blocked by protein synthesis inhibitor cycloheximide (CHX). FK506 was not able to mimic inhibitory effect of CsA on NO production in C6 cells, suggesting calcineurin-independent mechanism of CsA action.

Rat NKR-P1+ CD3+ T cells: selective proliferation in interleukin-2, diverse T-cell-receptor-Vbeta repertoire and polarized interferon-gamma expression.

Cells expressing markers of both natural killer and T lymphocytes (NK T cells) in humans and mice express a restricted T-cell receptor (TCR) repertoire, are of CD4- CD8- or CD4+ CD8- phenotype, and upon anti-CD3 stimulation secrete large amounts of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma). NK T cells may be the primary source of IL-4-promoting T helper type 2 (Th2) responses and/or they might be involved in regulating the balance between Th1- and Th2-type immune responses, and may consequently affect susceptibility to autoimmune diseases associated with a skewed Th phenotype. We show that rat NK T cells selectively proliferate to IL-2, and use this fact to analyse cytokine production by NK T cells in two rat strains differentially susceptible to Th1- or Th2-type autoimmune diseases. Analysis by reverse transcription-polymerase chain reaction revealed that, in contrast to mouse, rat NK T cells secrete exclusively IFN-gamma and not IL-4 after anti-CD3 stimulation, and use a wider TCR-Vbeta repertoire, suggesting that rat NK T cells are not essential for the development of Th2-type CD4+ T-cell responses.

Interleukin-1 receptor antagonist suppresses experimental autoimmune encephalomyelitis (EAE) in rats by influencing the activation and proliferation of encephalitogenic cells.

Considering the role of pleiotropic interleukin-1 (IL-1) in inflammation and autoimmunity, studies were designed to examine whether specific blockade of IL-1 may influence these processes in the CNS. Although the role of CD4+ T cells in eliciting clinical signs of experimental autoimmune encephalomyelitis (EAE) has been unequivocally demonstrated, the exact mechanism by which encephalitogenic cells initiate disease process and bring about clinical signs still remains to be defined. We have evaluated the effect of human recombinant interleukin-1 receptor antagonist (IL-1Ra) in vivo on the course of actively induced EAE in highly susceptible Dark Agouti (DA) rats. The rats which were treated during the induction phase of disease (days 0-6) with IL-1Ra (350 microg/rat/day) developed milder signs of EAE, when compared to saline-treated control animals immunized with encephalitogen, which developed severe single episode disease. The transfer of lymph node cells (LNC) isolated from MBP-primed DA rats and stimulated in vitro with MBP and ConA to naive syngeneic animals resulted in the development of EAE in all recipients. However, rats injected with LNC that have been stimulated in vitro in the presence of IL-1Ra (10 microg/ml) developed significantly milder disease. Diminished encephalitogenic capacity of LNC correlated with lower proliferative response to antigen and mitogen and decreased expression of IL-2 receptors. These data provide further evidence that IL-1 is an important factor for activation of EAE inducing T lymphocytes.

Pentoxifylline potentiates nitric oxide production and growth suppression in interferon-gamma-treated L929 fibroblasts.

In this study the effects of the phosphodiesterase inhibitor pentoxifylline (PTX) on nitric oxide (NO) production and proliferation of murine fibrosarcoma cell line L929 were investigated. We showed that both IFN-gamma (200 U/ml)-induced NO production and inhibition of [3H]thymidine uptake by L929 cells were potentiated in a synergistic fashion in the presence of PTX (200 micrograms/ml). These effects of PTX could be a consequence of phosphodiesterase inhibition, since they were mimicked by cAMP analogue dibutyryl cAMP. PTX failed to affect NO production when added to cells in which inducible NO synthase (iNOS) had already been induced with IFN-gamma and any further induction was blocked by cycloheximide (1 ug/ml), indicating that PTX modulates NO synthesis in L929 cells probably on a pretranslational level. Inhibition of iNOS with L-NAME (3 mM), although completely abolishing NO production, did not have any effect on proliferation of IFN-gamma or IFN-gamma + PTX-treated L929 cells, arguing against the possibility that growth suppression of these cells was due to the enhanced NO production. Moreover, the observation that the NO donor sodium nitroprusside (10-250 microM) significantly increased incorporation of [3H]thymidine in L929 fibroblasts suggests a role for NO in the positive regulation of their growth.

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression.

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.