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IL-10-induced DC (DC10) secrete IL-10, thereby promoting aeroallergen tolerance by activation of CD25+Foxp3+ Treg, while retinoic acid-induced DC (DC-RA) foster food allergen tolerance via IL-27-dependent induction of Foxp3− Treg. In some respects, these outcomes reflect those seen with aero- or food allergen-presenting lung and intestinal DC, respectively. Herein we asked whether these DCreg would also be functional in their reciprocal settings. DC-RA expressed lower levels of CCR5, CCR9, and CD103 than DC10, but higher levels of CD40, CD86, MHC II, TGF-β, IL-27 and Aldh1A2. DC-RA were also more effective in suppressing OVA-specific T cell proliferation in vitro (p ≤ 0.05). Co-culture of DC10 or DC-RA with OVA-specific T cells activated a regulatory phenotype therein, with the DC-10-induced Treg being modestly more suppressive in vitro than the DC-RA-induced Treg. We previously reported on lung trafficking of DC10, but DC-RA also traffic through the lungs and mediastinal lymph nodes before accumulating in the mesenteric lymph nodes. Both DCreg populations equally reduced airway hyperresponsiveness, Th2 responses (Th2 cytokines, eosinophilia) to airway allergen challenge, and plasma IgE/IgG1 levels in an OVA-asthma model. Similarly, they were equally effective in our OVA-food allergy model, where they reduced clinical scores (p ≤ 0.001), mast cell activation (p ≤ 0.05) and Th2 cytokine responses to allergen challenge, as well as OVA-specific IgE/IgG1 levels (p ≤ 0.01). Taken together, this data indicates that while DC10 and DC-RA employ distinct operative mechanisms, they were both able to induce tolerance in lung- and gut-associated allergic disease.

Dendritic cells (DC) are antigen-presenting cells that can communicate with T cells both directly and indirectly, regulating our adaptive immune responses against environmental and self-antigens. Under some microenvironmental conditions DC develop into anti-inflammatory cells which can induce immunologic tolerance. A substantial body of literature has confirmed that in such settings regulatory DC (DCreg) induce T cell tolerance by suppression of effector T cells as well as by induction of regulatory T cells (Treg). Many in vitro studies have been undertaken with human DCreg which, as a surrogate marker of antigen-specific tolerogenic potential, only poorly activate allogeneic T cell responses. Fewer studies have addressed the abilities of, or mechanisms by which these human DCreg suppress autologous effector T cell responses and induce infectious tolerance-promoting Treg responses. Moreover, the agents and properties that render DC as tolerogenic are many and varied, as are the cells’ relative regulatory activities and mechanisms of action. Herein we review the most current human and, where gaps exist, murine DCreg literature that addresses the cellular and molecular biology of these cells. We also address the clinical relevance of human DCreg, highlighting the outcomes of pre-clinical mouse and non-human primate studies and early phase clinical trials that have been undertaken, as well as the impact of innate immune receptors and symbiotic microbial signaling on the immunobiology of DCreg.

Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1—revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1—to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

CD40 expressed on stimulatory dendritic cells (DC) provides an important accessory signal for induction of effector T cell responses. It is also expressed at lower levels on regulatory DC (DCreg), but there is little evidence that CD40 signaling contributes to the tolerogenic activity of these cells. Indeed, CD40 silencing within DCreg has been reported to induce T cell tolerance in multiple disease models, suggesting that CD40 is superfluous to DC-induced tolerance. We critically assessed whether CD40 does have a role in tolerance induced by IL-10-differentiated DC (DC10) by using DC10 generating from the bone marrow of wild-type (w.t.) or CD40-/- donor mice, or IL-10-complemented CD40-/- DC10 to treat asthmatic mice. Wild-type DC10 ablated the OVA-asthma phenotype via induction of Foxp3+ Treg responses, but CD40-/- DC10 had no discernible effects on primary facets of the phenotype (e.g., IL-5, IL-9, IL-13 levels, IgE & IgG1 antibodies; p>0.05) and were ≤40% effective in reversal of others. Foxp3+ T cells from the lungs of CD40-/- DC10-treated mice expressed reduced levels of a panel of six Treg-specific activation markers relative to Treg from w.t. DC10-treated mice. Coculture with effector T cells from asthmatic mice induced a marked upregulation of cell surface CD40 on w.t. DC10. While untreated CD40-/- and w.t. DC10 secreted equally low levels of IL-10, stimulation of w.t. DC10 with anti-CD40 for 72 h increased their expression of IL-10 by ≈250%, with no parallel induction of IL-12. Complementing IL-10 expression in CD40-/- DC10 by IL-10 mRNA transfection fully restored the cells’ abilities to suppress the asthma phenotype. In summary, CD40 signaling in DC10 contributes importantly to their expression of IL-10 and to a robust induction of tolerance, including activation of induced Treg.

The cellular uptake, intracellular processing, and presentation of foreign antigen are crucial processes for eliciting an effective adaptive host response to the majority of pathogens. The effective recognition of antigen by T cells requires that it is first processed and then presented on MHC molecules that are expressed on other cells. A critical step leading to the presentation of antigen is delivering the foreign cargo to an intracellular compartment where the antigen can be processed and loaded onto MHC molecules. Fc-gamma receptors (FcγRs) recognize IgG-coated targets, such as opsonized pathogens or immune complexes (ICs). Cross-linking leads to internalization of the cargo with associated activation of down-stream signaling cascades. FcγRs vary in their affinity for IgG and intracellular trafficking, and therefore have an opportunity to regulate antigen presentation by controlling the shuttling and processing of their cargos. In this way, they critically influence physiological and pathophysiological adaptive immune cell functions. In this review, we will cover the contribution of FcγRs to antigen-presentation with a focus on the intracellular trafficking of IgG-ICs and the pathways that support this function. We will also discuss genetic evidence linking FcγR biology to immune cell activation and autoimmune processes as exemplified by systemic lupus erythematosus (SLE).

The prevalence of gout is relatively high worldwide, and many gout patients suffer from uric acid nephropathy (UAN) concomitantly. ELR-CXC chemokines such as CXCL8 and CXCL1 have a elevated expression in UAN. In this research, a mouse UAN model was established for a 12 week duration, and uric acid-related crystals were observed. CXCL8(3-72)K11R/G31P (G31P) is a mutant protein of CXCL8/interleukin 8 (IL-8), which has been reported to have therapeutic efficacy in both inflammatory diseases and malignancies for it acts as a selective antagonist towards CXCR1/CXCR2. In this study, G31P-treated mice showed declined production of the blood urea nitrogen (BUN) level and urine volume in UAN mice compared with G31P-untreated UAN counterparts. In addition, G31P effectively improved renal fibrosis, and reduced uric acid accumulation and leukocyte infiltration in UAN kidneys. Furthermore, the expressions of CXCL1 and CXCL2 were reduced and the activation of NOD-like receptors protein 3 (NLRP3) was inhibited by G31P treatment. This study has demonstrated that G31P attenuates inflammatory progression in chronic UAN, and plays a renoprotective function.

Calcitriol (1,25-dihydroxyvitamin D3, 1,25D3) and vitamin D side-chain modified analogs (VDAs) have gained considerable attention as potential drugs in the treatment of acute myeloid leukemia (AML), yet studies of the impact of 1,25D3 and VDAs upon other haematological malignancies are more limited. To address this gap in knowledge, we have examined the action of 1,25D3 and VDAs on a human cell line (DOHH2, K422) typifying diffuse large B-cell lymphoma (DLBCL) and also peripheral blood B-cells isolated from healthy donors. 1,25D3 and certain VDAs displayed moderate cytotoxic and pro-apoptotic actions upon DLBCL cells. 1,25D3 and VDAs (100nM) caused the death of approximately 40% DOHH2 cells after 24h stimulation, similar to their impact on HL-60 cells (acute myeloid leukaemia cell line). In addition, 1,25D3 and VDAs displayed concentration and time-dependent anti-proliferative actions upon stimulated B-cells from healthy donors. The VDAs inhibited proliferation by approximately 30%. Hence VDAs may offer therapeutic potential for the treatment of DLBCL or conditions benefitted by B-cell depletion.

Influenza during pregnancy is associated with the development of psychopathology in the offspring. We sought to determine whether maternal cytokines produced following administration of viral mimetic polyinosinic-polycytidylic acid (polyI:C) to pregnant rats were predictive of behavioral abnormalities in the adult offspring. Timed-pregnant Sprague Dawley rats received a single intravenous injection of 4-mg/kg polyI:C or saline on gestational day (GD)15. Blood was collected 3 h later for serum analysis of cytokine levels with ELISA. Male offspring were tested in a battery of behavioral tests during adulthood and behavior was correlated with maternal cytokine levels. Maternal serum levels of CXCL1 and interleukin (IL)-6, but not tumor necrosis factor (TNF)-α or CXCL2, were elevated in polyI:C-treated dams. PolyI:C-treated dams experienced post-treatment weight loss and polyI:C pups were smaller than controls at postnatal day (PND)1. Various behavior alterations were seen in the polyI:C-treated offspring. Male polyI:C offspring had enhanced MK-801-induced locomotion, and reduced sociability. PolyI:C offspring failed to display crossmodal and visual memory, and oddity preference was also impaired. Set-shifting, assessed with a lever-based operant conditioning task, was facilitated while touchscreen-based reversal learning was impaired. Correlations were found between maternal serum concentrations of CXCL1, acute maternal temperature and body weight changes, neonatal pup mass, and odd object discrimination and social behavior. Overall, while the offspring of polyI:C-treated rats displayed behavior abnormalities, maternal serum cytokines were not related to the long-term behavior changes in the offspring. Maternal sickness effects and neonatal pup size may be better indicators of later effects of maternal inflammation in the offspring.

Inflammation-related cerebral damage mediated by infiltrating neutrophils following reperfusion plays a role in reperfusion-induced brain damage subsequent to a stroke event. The ELR-CXC family of chemokines are CXCR1 and CXCR2 agonists that are known to drive neutrophil migration and activation. The present study demonstrated the benefit of anti-inflammatory therapy in the treatment of ischemic stroke with the administration of the competitive ELR-CXC chemokine antagonist, CXCL8(3-72)K11R/G31P (G31P). Male Sprague-Dawley rats were anaesthetized, and the middle cerebral artery (MCA) was occluded for 30 min followed by 5.5 h of reperfusion. Pretreatment with G31P resulted in a significant, dose-dependent (approximately 61-72%) decrease in infarct volumes compared to vehicle-treated animals, but neuroprotection was also observed when G31P (0.5 mg/kg) was administered 1 or 3 h following the start of reperfusion. The neuroprotection observed following the administration of this competitive CXCR1/CXCR2 antagonist may present therapeutic opportunities for addressing reperfusion-induced inflammatory damage in patients presenting with transient ischemic episodes.

Research suggests that maternal immune activation (MIA) during pregnancy increases the risk of neurodevelopmental disorders including schizophrenia and autism in the offspring. Current theories suggest that inflammatory mediators including cytokines and chemokines may underlie the increased risk of these disorders in humans. For example, elevated maternal interleukin-8 (IL-8) during pregnancy is associated with increased risk of schizophrenia in the offspring. Given this association, the present experiments examined ELR-CXC chemokines CXCL1 and CXCL2, rodent homologues of human IL-8, and activation of their receptors (CXCR1 and CXCR2) in an established rodent model of MIA. Pregnant Long Evans rats were treated with the viral mimetic polyinosinic–polycytidylic acid (polyI:C; 4 mg/kg, i.v.) on gestational day 15. Protein analysis using multiplex assays and ELISA showed that polyI:C significantly increased maternal serum concentrations of interleukin-1β, tumor necrosis factor, and CXCL1 3 h after administration. Subsequent experiments tested the role of elevated maternal CXCL1 on behavior of the offspring by administering a CXCR1/CXCR2 antagonist (G31P; 500 μg/kg, i.p.; 1 h before, 48 and 96 h after polyI:C treatment). The male offspring of dams treated with polyI:C demonstrated subtle impairments in prepulse inhibition (PPI), impaired associative and crossmodal recognition memory, and altered behavioral flexibility in an operant test battery. While G31P did not completely reverse the behavioral impairments caused by polyI:C, it enhanced PPI during adolescence and strategy set-shifting and reversal learning during young adulthood. These results suggest that while polyI:C treatment significantly increases maternal CXCL1, elevations of this chemokine are not solely responsible for the effects of polyI:C on the behavior of the offspring.

The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), the expression levels of which are elevated in many inflammatory diseases, binds to both the CXCR1 and CXCR2 receptors with high affinity. Recently, an analogue of human CXCL8, CXCL8(3–72)K11R/G31P (hG31P) has been developed. It has been demonstrated that hG31P is a high affinity antagonist for both the CXCR1 and CXCR2. Herein, we have determined the solution structure and the CXCR1 N-terminal peptide binding sites of hG31P by NMR spectroscopy. We have found that the displacement within the tertiary structure of the 30 s loop and the N-terminal region and more specifically change of the loop conformation (especially H33), of hG31P may affect its binding to the CXCR1 receptor and thereby inhibit human neutrophil chemotactic responses induced by ELR-CXC chemokines. Our results provide a structural basis for future clinical investigations of this CXCR1/CXCR2 receptor antagonist and for the further development of CXCL8 based antagonists.

Klebsiella pneumoniae-associated pathology is largely mediated by neutrophilic inflammation. In this study, we administeredKlebsiella pneumoniaeto experimental guinea pig groups and the ELR-CXC chemokine antagonistCXCL83–72, ceftazidime, and dexamethasone to different groups, respectively. After 24 h, we assessed the animal’s pulmonary inflammatory levels, including gross histopathology, airway neutrophilia, lung myeloperoxidase levels, expressions of CXCL8 and TNF, and airway bacterial loads. Compared with ceftazidime and dexamethasone treatments, the administration of the ELR-CXC chemokine antagonistCXCL83–72alone was more effective than other methods, although it did not markedly attenuate the bacterial load. These results suggest new methods for the treatment ofKlebsiella pneumoniaepathology.

IL-10–differentiated dendritic cells (DC10s) can prevent allergen sensitization and reverse the asthma phenotype in mice with established disease. However, little is known about the time-frames over which this tolerance is effective. We report that at 2 wk after i.p. or transtracheal delivery of 1 × 106 OVA-, but not house dust mite- presenting, DC10s to OVA-asthmatic mice, significant diminution of airway hyperresponsiveness (AHR) was first apparent, whereas AHR was abrogated between 3 and 10 wk posttreatment. At 13 wk, AHR returned to pretreatment levels but could again be reversed by DC10 retreatment. The impact of a single DC10 treatment on airway eosinophil and Th2 cytokine responses to recall OVA challenge, and on OVA-specific IgE/IgG1 responses, was substantial at 3 wk posttreatment, but progressively increased thereafter, such that at 8 mo, airway eosinophil and Th2 responses to recall allergen challenge remained ∼85–95% suppressed relative to saline-treated asthmatic mice. Four biweekly DC10 treatments, whether transtracheal or i.p., reduced all asthma parameters to near background by 8 wk, whereas s.c. DC10 treatments did not affect AHR but did reduce the airway Th2 responses (i.v. DC10 had no discernible effects). Repeated challenge of the DC10-treated mice with aerosolized OVA (100 μg/ml) did not reverse tolerance, but treatment with the indoleamine-2,3-dioxygenase antagonist 1-methyltryptophan or neutralizing anti–IL-10R from days 12 to 21 after DC10 therapy partially reversed tolerance (Th2 cytokine responses, but not AHR). These findings indicate that DC10-induced Th2 tolerance in asthmatic animals is long lived, but that DC10s employ distinct mechanisms to affect AHR versus Th2 immunoinflammatory parameters.

Prostate cancer is the most common malignancy in Western countries. Chemokine C-X-C motif receptor 1 (CXCR1) and CXCR2 play a key role in generation and regulation of CXC chemokine signaling. CXCR1 is a receptor for interleukin 8 (IL8), a pro-inflammatory chemokine, and CXCR1/2 are crucially involved in the prostate cancer development and progression. Thus, we generated a high-affinity human CXCR1/CXCR2 inhibitor, CXCL8 (3-72) K11R/G31P, named G31P, which is a synthetic derivative of the human cytokine, IL-8. In this study, we investigated the effects of G31P on regulation of prostate cancer cell growth in vitro and in nude mouse xenografts. Cell viability, adhesion, and wound healing assays were used to assess the effects of G31P on growth, adhesion, and migration of PC-3 human prostate cancer cells in vitro, respectively. Nude mouse xenografts and xenograft implantation assays were performed to determine the effect of G31P on PC-3 cells in vivo. Immunohistochemistry was used to detect gene expression, and fluorescence imaging was used to detect tumor volume and microvessel density in tumor xenografts. The data showed that G31P treatment significantly reduced PC-3 cell viability, adhesion and migration capacity in a dose-dependent manner (up to 100 ng/ml). Additionally, G31P treatment of nude mice suppressed the growth of orthotopically transplanted tumor xenografts. G31P also inhibited tumor tissue vascularization, which was associated with the decreased expression of vascular endothelial growth factor and nuclear transcription factor (NF)-κB in orthotopic xenograft tissues. This study provides evidence that G31P, a CXCR1/2 inhibitor, may effectively control prostate cancer.

Immune modulation has been a sought after means of therapy for atopic diseases. CVT-E002 is an extract derived from North American Ginseng shown to promote T-helper-1-like responses. We determined what effect CVT-E002 could have in a mouse model of atopic asthma. We report that oral CVT-E002 inhibited the development of allergic airway inflammation and airway hyperresponsiveness. This correlated with an increased presence of interferon-γ in the lung, and also increased regulatory T cells and IL-10. The ability of CVT-E002 to induce regulatory T-cell development was also seen in human in vitro co-cultures.

We have shown that proteinase-activated receptor-2 (PAR2) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR2 in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR2. To study the role of PAR2 in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR2 activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR2, mice were exposed intranasally to a receptor-blocking anti-PAR2 Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR2 blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR2 activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR2-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR2 activation may be a general mechanism used by aeroallergens to induce allergic sensitization.

To cite this article: Lu M, Dawicki W, Zhang X, Huang H, Nayyar A, Gordon JR. Therapeutic induction of tolerance by IL-10-differentiated dendritic cells in a mouse model of house dust mite-asthma. Allergy 2011; 66: 612–620. Abstract Background: It has been reported that retrovirally transduced IL-10-expressing dendritic cells can reverse the asthma phenotype in mice, but that i.v. delivery of dendritic cells differentiated with IL-10 alone (DC10) does not. We report herein DC10 can be highly effective therapeutically in experimental asthma. Methods: BALB/c mice were sensitized by airway exposure to house dust mite (HDM) without use of adjuvants, then treated with 106 allergen-presenting DC10. We assessed the airway hyperresponsiveness (AHR) to methacholine, circulating levels of IgE and IgG1, and airway recall responses to HDM allergen, including eosinophilia and Th2 cytokines. We also asked whether the DC10 treatments induced tolerance through activation of pulmonary regulatory T cell activities. Results: In vitro, cognate-, but not irrelevant-, allergen-presenting DC10 productively engaged pulmonary Th2-phenotype CD4+ cells magnetically sorted from HDM-asthmatic mice in Forster (or fluorescence) resonance energy transfer assays. In vivo, treatment of HDM-asthmatic mice with HDM, but not ovalbumin-presenting DC10 abrogated AHR within 4 weeks, and significantly reduced airway eosinophilia, IL-4, IL-5, and IL-13 responses, and circulating HDM-specific IgE and IgG1 levels (each, P ≤ 0.01 versus control mice). CD4+CD25+Foxp3+ cells from the lungs of the DC10-treated mice, but not those from asthmatic animals, up-regulated expression of the activated regulatory T cell markers CTLA4 and LAG3, and passive transfer of pulmonary CD4+ T cells from these mice induced allergen tolerance in HDM-asthmatic recipients. Conclusions: These findings indicate that allergen-presenting DC10 treatments up-regulate T cell regulatory activities and thereby induce allergen-specific tolerance in a relevant model of human asthma.

IL-10–differentiated dendritic cells (DC10) induce allergen tolerance in asthmatic mice, during which their lung Th2 effector T cells (Teffs) are displaced by activated CD4+CD25hiFoxp3+ T cells. Intestinal DCs promote oral tolerance by inducing Ag-naive T cells to differentiate into CD4+CD25+Foxp3+ regulatory T cells (Tregs), but whether DCs can induce Teffs to differentiate into Tregs remains uncertain. In this study, we addressed this question in OVA-asthmatic mice that were treated with DC10. OVA-presenting DC10 treatment maximally activated lung Tregs in these animals at 3 wk posttreatment, as determined by upregulation of activation markers (ICOS, programmed cell death-1, glucocorticoid-induced TNFR-related protein, LAG3, and CTLA-4) and in functional assays. This in vitro regulatory activity was ≥90% reduced by treatment with anti–IL-10 but not anti–TGF-β Abs. In parallel cultures, OVA- but not house dust mite (HDM)-presenting DC10 induced ≈43% of CFSE-labeled CD25−/loFoxp3− Teffs from asthmatic OVA–TCR transgenic mice to differentiate into tolerogenic CD25hiFoxp3+ Tregs. We recapitulated this in vivo using OVA-asthmatic mice that were coinjected with OVA- or HDM-presenting DC10 (i.p.) and CFSE-labeled CD4+CD25-/loFoxp3− Teffs (i.v.) from the lungs of asthmatic DO11.10 mice. From ≈7 to 21% of the activated (i.e., dividing) DO11.10 Teffs that were recovered from the lungs, lung-draining lymph nodes, or spleens of the OVA–DC10 recipients had differentiated into CD4+CD25hiFoxp3+ Tregs, whereas no CFSE-positive Tregs were recovered from the HDM–DC10-treated animals. These data indicate that DC10 treatments induce tolerance at least in part by inducing Teffs to differentiate into CD4+CD25hiFoxp3+ Tregs.

Background Neutrophil sequestration plays an important role in mediating local and remote organ injury induced by ischemia and reperfusion (I/R). The Glu-Leu-Arg (ELR)-CXC subfamily of chemokines, all CXCR1 or CXCR2 ligands, are primary agonists for such neutrophil recruitment. Herein, we assessed the effects of a combined CXCR1/CXCR2 antagonist, CXCL8 (3-72) K11R/G31P (G31P), on neutrophilic local (gut) and distant organ injury and outcomes after superior mesenteric artery I/R in rats. Methods Male Sprague-Dawley rats ( n = 6–10) were subjected to either sham treatment or superior mesenteric artery ischemia for 1 h; all animals received either saline or G31P (500 μg/kg, s.c.) and were euthanized for assessment after either 2 or 5 h of arterial reperfusion. Survival and gut pathology, and pulmonary neutrophils were assessed directly, while bronchoalveolar lavage (BAL) fluid total protein levels and red blood cell (RBC) numbers were determined by protein assay and direct counting. Expression of inflammatory mediators in the lung and jejunum was measured by quantitative RT-PCR, colorimetric or gel zymography assays. Results Sham treatment animals suffered no discernible gut or pulmonary pathology. At 2 and 5 h after reperfusion, the survival levels of the saline-treated I/R injury animals were 80% and 50%, respectively, while all G31P-treated animals survived. I/R injury led to substantial villous pathology within the jejunum, and G31P significantly reduced these pathology scores as well as neutrophil infiltration of the jejunal lamina propria and lung parenchyma, and vascular leakage into the airways (BAL protein). The tissue injury increased expression of myeloperoxidase and matrix metalloproteinase (MMP)-2 and MMP-9 in the gut tissues, but G31P treatment did not significantly affect this response. Intestinal I/R increased expression of IL-1, IL-6, GRO, and MIP-2 in the ischemic jejunum and the lung tissues, but here too G31P treatment had no palliative effects on these responses. Conclusion These results suggest that full-spectrum ELR-CXC chemokine antagonism has significant protective effects against I/R-induced local and remote organ injury.

In mouse models of asthma, therapeutic use of allergen-presenting IL-10-differentiated dendritic cells (DCs) can abrogate airway hyperresponsiveness, and reduce other asthma-related responses to near background. Analogous human DCs can suppress human T cell responses in vitro, but the operative mechanisms are poorly defined. We investigated the ability of IL-10-treated human DCs to induce tolerance among autologous T cells of subjects with asthma and the mechanisms by which they do this. CD14(+) monocyte-derived DCs were differentiated in the presence of IL-10 (DC10) ex vivo from 11 donors with asthma and 4 control donors, and characterized for relevant markers. They were pulsed with specific or irrelevant allergen, and cultured with autologous peripheral blood CD4(+) T cells, either alone or together with autologous immunostimulatory DCs (DC-TNF), and the impact of this treatment on the T-cell responses was assessed for each donor. The DC10 expressed reduced levels of some relevant markers (CD40, CD80, human leukocyte antigen-DR) and stimulatory cytokines (IL-6 and IL-12), but augmented levels of Ig-like transcript-22/CD85j and IL-10 relative to DC-TNF. In cocultures, they dampened DC-TNF-driven T helper (Th) type 2 cell proliferation and cytokine (IL-4, -5, and -13) secretion. They also drove the development from atopic CD4(+)CD25(lo)Foxp3(lo) cells of a population of IL-10-secreting CD25(+)Foxp3(+)LAG-3(+)CTLA-4(+) regulatory T cells (Tregs). These Tregs suppressed stimulatory DC-induced autologous Th2 cell proliferation and cytokine secretion in a contact-dependent manner. Our data indicate that IL-10-treated human DCs induce Th2 cell allergen tolerance ex vivo by driving the differentiation of Tregs.

Pneumonia associated with aspiration of bacterial-laden gastric contents is characterized by Glu-Leu-Arg (ELR)-CXC chemokine (e.g., CXC2L1, CXCL8) expression leading to local neutrophil sequestration. This neutrophil response is designed to be protective, but overly aggressive responses can be pathogenic in themselves. Herein we assessed whether blocking neutrophil responses in a guinea pig model of aspiration pneumonia would foster airway bacterial growth. Guinea pigs (n=5) were challenged intranasally with saline, acidified saline or acidified gastric contents (35mg/kg body weight, pH 2.0) and treated subcutaneously with 250mug/kg of the human ELR-CXC chemokine antagonist CXCL8((3-72))K11R/G31P (G31P) or saline. After 20h the animals' airway inflammatory responses and bacterial burdens were assessed. A loss of vascular integrity was apparent in the lungs of the saline-treated aspiration pneumonia animals (12.07+/-1.3% of the pleural surfaces exhibited hemorrhagic consolidation, 4.6x10(6) RBC/ml bronchoalveolar lavage fluid [BALF]), as was a pulmonary neutrophilia. The BAL fluids contained gram-negative and -positive bacteria (total load, 6.3+/-3.2x10(7) CFU/ml BALF) that are associated with nosocomial infections in humans. The G31P-treatments attenuated the pulmonary vascular complications (2.27+/-0.5% pleural surface hemorrhagic consolidation, 0.46x10(6) RBC/ml BALF), and reduced the pulmonary neutrophilia by >/=86%. The G31P-treatments did not lead to significant changes in the airway bacterial loads (total load, 3.46+/-1.8x10(7) CFU/ml BALF). This data indicates that attenuation of the pulmonary neutrophil response in aspiration pneumonia reduces pathology very significantly but does not reduce the efficiency of pulmonary bacterial clearance.

The ELR-CXC chemokines play important roles in neutrophilic inflammation. We report in this study that a fully human ELR-CXC chemokine antagonist that we have generated, CXCL8(3–72)K11R/G31P (G31P), has potent anti-inflammatory effects that arise through its actions at multiple levels. G31P inhibited CXCL8-induced chemotactic responses and intracellular Ca2+ flux in CXCR1-transfected HEK cells and neutrophils, and responses of neutrophils to CXCR2-exclusive ligands. G31P desensitized heterologous G protein-coupled receptors on neutrophils, 52–86% reducing their Ca2+ flux and chemotactic responses to leukotriene B4, C5a, and the bacterial tripeptide fMLP. G31P also 60–90% blocked neutrophil chemotactic responses to mediators present in 10 of 12 sputum samples from cystic fibrosis or bronchiectasis subjects with bacterial pneumonia. Moreover, whereas A549 bronchial epithelial cells (which expressed CXCR1) secreted ≈29,000 pg/ml CXCL8 in response to in vitro endotoxin challenge, G31P reduced this response by up to 98%, presumably by interrupting an autocrine inflammatory loop. The anti-inflammatory effects of G31P extended also to reversing the antiapoptotic influence of ELR-CXC chemokines on neutrophils. That these effects were relevant in vivo was confirmed in a guinea pig model of airway endotoxemia, wherein the human form of G31P >95% blocked neutrophil infiltration into and activation within the airways, as determined by airway levels of the neutrophil primary, secondary, and tertiary granule markers myeloperoxidase, lactoferrin, and matrix metalloproteinase-9, respectively, and the epithelial cell marker matrix metalloproteinase-2. These data suggest that the beneficial effects of ELR-CXC chemokine antagonism arise through effects that occur at multiple levels, including epithelial cells, neutrophils, and alternate G protein-coupled receptors.

Hepatitis C virus (HCV) primarily replicates within the liver, leading to hepatitis, fibrosis, and hepatocellular carcinoma. Infection is also associated with B-cell abnormalities, suggesting an association of the virus with B cells. The infectious JFH-1 strain of HCV can bind primary and immortalized B cells but fails to establish productive infection. However, B cell–associated virus readily infects hepatoma cells, showing an enhanced infectivity compared with extracellular virus. B cells express the viral receptors CD81, SR-BI, and the C-type lectins DC-SIGN and L-SIGN. Antibodies specific for SR-BI and DC-SIGN/L-SIGN reduced B-cell transinfection, supporting a role for these molecules in B-cell association with HCV. Stimulation of B cells with CD40 ligand and interleukin-4 promoted their ability to transinfect hepatoma cells. B cell–associated virus is resistant to trypsin proteolysis and HCV-specific neutralizing antibodies, consistent with particle internalization. HCV promoted the adhesion of primary B cells to Huh-7 hepatomas, providing a mechanism for B-cell retention in the infected liver. In summary, B cells may provide a vehicle for HCV to persist and transmit to the liver.

International audience; Benzo(a)pyrene (BP) is an environmental contaminant known to favor airway inflammation likely through up-regulation of pro-inflammatory cytokines. The present study was designed to characterize its effects toward interleukin-8 (IL-8), a well-established pulmonary inflammatory cytokine. In primary human macrophages, BP was shown to induce IL-8 expression at both mRNA and secretion levels in a dose-dependent manner. Such an up-regulation was likely linked to aryl hydrocarbon receptor (AhR)-activation since BP-mediated IL-8 induction was reduced after AhR expression knock-down through RNA interference. Moreover, electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation experiments showed BP-triggered binding of AhR to a consensus xenobiotic responsive element (XRE) found in the human IL-8 promoter. Finally, BP administration to mice led to over-expression of keratinocyte chemoattractant (KC), the murine functional homologue of IL-8, in lung. It also triggered the recruitment of neutrophils in bronchoalveolar lavage (BAL) fluids, which was however fully abolished in the presence of a chemical antagonist of the KC/IL-8 receptors CXCR1/CXCR2, thus supporting the functional and crucial involvement of KC in BP-induced lung inflammation. Overall, these data highlight an AhR-dependent regulation of IL-8 in response to BP that likely contributes to the airway inflammatory effects of this environmental chemical.

Glu-Leu-Arg (ELR)-CXC chemokines are important in acute responses to bacterial infections, wherein neutrophils are often critical to pathogen clearance. However, excessive neutrophil recruitment augments the pathology of many diseases. We have shown that bovine CXCL8((3-74))K11R/G31P (bG31P) is a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle, but herein we wished to determine whether humanized forms of this antagonist would be similarly effective. We thus examined the independent contributions of the bovine-human-discrepant amino acids within CXCL8 to the biological activity of bG31P. We first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P, and found that this human-bovine chaemeric G31P (hbG31P; i.e., bCXCL8((3-44))K11R/G31P-hCXCL8((45-72))) fully retained the ELR-CXC chemokine antagonist activity of bG31P. Thus, hbG31P blocked the abilities of human CXCL8 to chemoattract human neutrophil or induce reactive oxygen intermediate (ROI) release. It was also a highly effective antagonist in vivo in a guinea pig model of airway endotoxemia. We next methodically moved through the 5' half of the hbG31P cDNA, using site-directed mutagenesis to one-by-one make substitutions at each remaining discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T). We generated and tested the agonist and antagonist activities of each analogue using human neutrophils and human CXCL8. We found that none of these possessed better antagonist activities than hbG31P. Our data thus suggests that partially humanized analogues of bG31P display significant potential as antagonists of human ELR-CXC chemokines.

Human B lymphocytes and derived lines from a spectrum of B cell malignancy were studied for expression of dopaminergic pathway components and for their cytostatic response to the catecholamine and related, potentially therapeutic compounds. Proliferating normal lymphocytes and dividing malignant clones rapidly arrested on exposure to dopamine in the low (≤10 μM) micromolar range. The antiparkinsonian drugs l -DOPA and apomorphine (particularly) were similarly antiproliferative. With the exception of D4, dopamine receptors D1–D5 were variably expressed among normal and neoplastic B cell populations, as was the dopamine transporter. Transcripts for D1 and D2 were frequently found, whereas D3 and D5 revealed restricted expression; dopamine transporter was detected in most cases. Nevertheless, pharmacological analysis disclosed that dopamine targeted cycling B cells independent of these structures. Rather, oxidative stress constituted the primary mechanism: the catecholamine’s actions being mimicked by hydrogen peroxide and reversed by exogenous catalase, and evidence for the intracellular redox protein thioredoxin contributing protection. Among proliferating clones, growth arrest was accompanied by cell death in populations deplete in antiapoptotic Bcl-2: resting lymphocytes escaping low micromolar dopamine toxicity. Dysregulated bcl-2 expression, although preventing oxidative-induced caspase-dependent apoptosis, by itself conferred only minor protection against dopamine cytostasis. The selective impact of dopamine on lymphocytes that are in active cycle indicates an axis for therapeutic intervention not only in B cell neoplasia but also in lymphoproliferative disturbances generally. Rational tailoring of drug delivery systems already in development for Parkinson’s disease could provide ideal vehicles for carrying the oxidative hit directly to the target populations.

We previously showed that the human AKNA gene encodes an AT-hook transcription factor that regulates the expression of costimulatory cell surface molecules on lymphocytes. However, AKNA cDNA probes hybridize with multiple transcripts, suggesting either the existence of other homologous genes or a complex regulation operating on a single gene. Here we report evidence for the latter, as we find that AKNA is encoded by a single gene that spans a 61-kb locus of 24 exons on the fragile FRA9E region of human chromosome 9q32. This gene gives rise to at least nine distinct transcripts, most of which are expressed in a tissue-specific manner in lymphoid organs. Many of the AKNA transcripts originate from alternative splicing; others appear to derive from differential polyadenylation and promoter usage. The alternative AKNA transcripts are predicted to encode overlapping protein isoforms, some of which (p70 and p100) are readily detectable using a polyclonal anti-AKNA antisera that we generated. We also find that AKNA PEST-dependent cleavage into p50 polypeptides is targeted to mature B cells and appears to be required for CD40 upregulation. The unusual capacity of the AKNA gene to generate multiple transcripts and proteins may reflect its functional diversity, and it may also provide a fail-safe mechanism that preserves AKNA expression.

Background Serine proteinases such as mast cell tryptase, trypsin-like enzymes, and certain allergens are important in the pathogenesis of asthma. These proteinases can activate the proteinase-activated receptor (PAR)–2, which has been shown to be upregulated in the airways of patients with asthma. Objective The purpose of this study was to investigate PAR-2 activation in the airways during allergen challenge and its effects on the 2 principle features of asthma, airway inflammation and airway hyperresponsiveness (AHR). Methods Proteinase-activated receptor 2 activating peptide SLIGRL-NH2 (PAR-2 activating peptide [ap]) or control peptide LSIGRL-NH2 (PAR-2 control peptide [cp]) was administered alone or in conjunction with ovalbumin intranasally to mice, and AHR and airway inflammation were evaluated. Results PAR2ap did not induce AHR or airway inflammation in ovalbumin-sensitized mice that had not been challenged with ovalbumin. When administered with ovalbumin, PAR-2ap enhanced AHR and airway inflammation compared with ovalbumin administered alone or with PAR-2cp. The enhanced AHR persisted for 5 days, whereas the enhancement to airway inflammation dissipated. Mice administered PAR-2ap alone during the 5 days after the final antigen challenge demonstrated an additional enhancement to airway inflammation compared with the control animals. PAR-2ap administered with allergen increased TNF and IL-5 mRNA in lung tissue and IL-13 and TNF in bronchoalveolar lavage fluid. Conclusion Exogenous PAR-2 activation in parallel with allergen challenge enhances allergen-mediated AHR and airway inflammation through distinct mechanisms. PAR-2 activation can also enhance established airway inflammation even when dissociated from exposure to allergen. Therefore, PAR-2 activation may play a pathogenic role in the development of AHR and airway inflammation.

Regular salbutamol use can exacerbate allergen-induced airway eosinophilia in asthmatics, but its effect on airway eosinophil chemokine responses is unknown. Asthmatic subjects (n=14) were treated for 10 days with placebo or salbutamol in a double-blind, cross-over study, then given same-dose allergen challenges. Their sputa were then analysed 1 and 7 h later for a panel of eosinophil-related cytokines. Eosinophils from five test and three control subjects were tested for expression of CXCL8/interleukin (IL)-8, and its receptors and responsiveness to CCL11/eotaxin and CXCL8/IL-8. Sputum CXCL8/IL-8, but not IL-5, CCL5/regulated on activation, T-cell expressed and secreted, CCL7/monocyte chemotactic protein-3, CCL11/eotaxin, granulocyte-macrophage colony-stimulating factor or tumour necrosis factor levels, were increased (42%) by the salbutamol treatments. The CXCL8/IL-8 levels correlated with the proportions of sputum eosinophils and these cells, but not other sputum cells, stained strongly for CXCL8/IL-8. The circulating eosinophils of the tested subjects (n=5) expressed CXCL8/IL-8 receptors and secreted high levels of this chemokine. Neutralisation of sputum CXCL8/IL-8 reduced eosinophil chemotactic responses to these samples by 19 +/- 5%. These data suggest that regular use of salbutamol can augment airway CXCL8/interleukin-8 responses to allergen challenge and that this CXCL8/interleukin-8 could contribute to the airway inflammatory response.

Tumour antigen presentation by dendritic cells (DCs) to T cells in lymphoid organs is crucial for induction of anti-tumour immune responses. It has been previously reported that tumour necrosis factor-alpha (TNF-alpha) is required for DC activation and subsequent induction of optimal immune responses, and thus DCs for anti-tumour vaccination are often generated by culture in exogenous TNF-alpha. In the present study, we investigated the effect on anti-tumour immunity of vaccination with Mut1 tumour peptide-pulsed DCs engineered to express a TNF-alpha transgene. Our data shows that transfection of DCs with recombinant adenovirus AdV-TNF-alpha resulted in greater maturation of the DCs than occurred with control DCs cultured in exogenous TNF-alpha, as determined by up-regulated expression of pro-inflammatory cytokines (e.g. interleukins 1beta and 18), chemokines [e.g. interferon-gamma-inducible protein-10 and macrophage inflammatory protein-1beta (MIP-1beta)], the CC chemokine receptor CCR7, and immunologically important cell surface molecules (CD40, CD86 and intercellular adhesion molecule-1). These transgenic DCs stimulated stronger allogeneic T-cell responses in vitro and T-cell activation in vivo; displayed 2.4-fold enhanced chemotactic responses to the MIP-3betain vitro (P

IL-10-differentiated dendritic cells (DC10) can reverse the asthma phenotype in mice, but how they suppress the asthmatic B cell response is unclear. Herein we assessed the mechanism(s) by which DC10 and DC10-induced Treg affect IgG1 production in asthma. We observed a rapid decline in lung-resident OVA-specific IgG1-secreting B cells on cessation of airway allergen challenge, and intraperitoneal DC10 therapy did not amplify that (p>0.05). It did however increase the loss of IgG1-B cells from the bone marrow (by 45+/-7.2%; p≤0.01) and spleen (by 65+/-17.8%; p≤0.05) over 2 wk. Delivery of OVA-loaded DC10 directly into the airways of asthmatic mice decreased the lung IgG1 B cell response assessed 2 dy later by 33+/-9.7% (p≤0.01), while their co-culture with asthmatic lung cell suspensions reduced the numbers of IgG1-secreting cells by 56.5+/-9.7% (p≤0.01). This effect was dependent on the DC10 carrying intact allergen on their cell surface; DC10 that had phagocytosed and fully processed their allergen were unable to suppress B cell responses, although they did suppress asthmatic Th2 cell responses. We had shown that therapeutic delivery of DC10-induced Treg can effectively suppress asthmatic T and B cell (IgE and IgG1) responses; herein CD4+ cells or Treg from the lungs of DC10-treated OVA-asthmatic mice suppressed in vitro B cell IgG1 production by 52.2+/-8.7% (p≤0.001) or 44.6+/-12.2% (p≤0.05), respectively, but delivery of DC10-induced Treg directly into the airways of asthmatic mice had no discernible impact over 2 dy on the numbers of lung IgG1-secreting cells (p≥0.05). In summary, DC10 treatment down-regulates OVA-specific B cell responses of asthmatic mice. While DC10 that carry intact allergen on their cell surface can dampen this response, DC10-induced Treg are critical for full realization of this outcome. This suggests that infectious tolerance is an essential element in regulatory DC control of the B cell response in allergic asthma.

Supplemental data for this article can be accessed on the publisher’s website. © 2015 The Author(s) & Taylor & Francis Group, LLC. Rapamycin is a well-known inhibitor of the Target of Rapamycin (TOR) signaling cascade; however, the impact of this drug on global genome function and organization in normal primary cells is poorly understood. To explore this impact, we treated primary human foreskin fibroblasts with rapamycin and observed a decrease in cell proliferation without causing cell death. Upon rapamycin treatment chromosomes 18 and 10 were repositioned to a location similar to that of fibroblasts induced into quiescence by serum reduction. Although similar changes in positioning occurred, comparative transcriptome analyses demonstrated significant divergence in gene expression patterns between rapamycin-treated and quiescence-induced fibroblasts. Rapamycin treatment induced the upregulation of cytokine genes, including those from the Interleukin (IL)-6 signaling network, such as IL-8 and the Leukemia Inhibitory Factor (LIF), while quiescent fibroblasts demonstrated up-regulation of genes involved in the complement and coagulation cascade. In addition, genes significantly up-regulated by rapamycin treatment demonstrated increased promoter occupancy of the transcription factor Signal Transducer and Activator of Transcription 5A/B (STAT5A/B). In summary, we demonstrated that the treatment of fibroblasts with rapamycin decreased proliferation, caused chromosome territory repositioning and induced STAT5A/B-mediated changes in gene expression enriched for cytokines. Brunel Progeria Research Fund (UK), Canadian Institutes of Health Research, NSERC Discovery Grant, NSERC USRA program, Royal Society Research Grant (UK), University of Saskatchewan NSERC President’s Fund, Saskatchewan Innovation and Opportunity Fund.

T cells are a key element in effective cancer immunity, recognizing MHC-antigen peptide complexes on the surface of antigen presenting cells and translating these signals into cytotoxic effector T cell responses. In this study, we systematically investigated by DNA array analysis the expression profiles of 514 immunologically relevant genes in naïve and SP2/0 tumor-specific activated mouse T cell populations. Our data shows that naïve T cells expressed 37 (i.e., 7.6% of the 514) transcripts with expression level (EL) values ofor =2.0, while the activated T cells expressed 101 such transcripts. The expression levels of 9 (1.75% of 514) of the shared transcripts were equivalent in the two populations of T cells. Ninety-six genes were differently expressed upon T cell activation, with 71 (13.81%) being up-regulated and 25 (4.86%) down-regulated. The list of significantly affected genes includes numerous cytokines and their receptors (e.g., IL-2Ralpha, IL-6Ralpha, IL-7Ralpha, IL-16, IL-17R, TGF-beta), chemokines and chemokine receptors (e.g., RANTES, CCR7, CXCR4), alternate surface proteins (e.g., 4-1BB, GITR, integrins-alphaL and -beta7, L-selectin, CD6, CD45 and EMMPRIN), cytoplasmic signaling intermediates (e,g., GATA-3, 14-3-3-eta, CIS1, SMAD4 and JAK1) and an array of other molecules (e.g., NFkappa-B inducing kinase, LTBP3 and persephin), several of which are associated with Th1 responses, and T cell self-regulation or migration. Taken together, our data contribute to our understanding of the generalized processes that accompany T cell activation and, more specifically, to our understanding of the processes associated with T cell activation during antitumor responses.

Population size is governed through cells reacting to a variety of intrinsic and extrinsic cues. Tumors, while liberated from many of the homeostatic constraints placed on physiologic counterparts, can nonetheless remain subject to both social and environmental control. Burkitt lymphoma cells faithful to the biopsy phenotype were used to model the reliance of the colony, if any, on an inbuilt population sensor. Below a normally suicidal threshold number of cells, low picomolar quantities of exogenous CD40 ligand (CD40L/CD154) were found to sustain the clone without the discernible shift in phenotype that accompanies high CD40L encounter. Although CD154 was undetectable in populous cultures, message was induced as numbers became limiting. Correspondingly, attempts to neutralize endogenous CD40L activity failed to perturb cells at optimal densities but resulted in their marked decline as the critical threshold was approached. These data reveal an auto-inducible survival mechanism seemingly regulated through the monitoring of population size, a process somewhat akin to that of “quorum sensing” among gram-negative bacteria in which diffusible molecules provide a means of communication to coordinate gene expression with population density. This process could be activated as cells discern depletions in their community or when deprived of signals otherwise furnished within an appropriate environmental niche.

Serotonin (5-HT), a well-known neurotransmitter of the central nervous system, has been implicated in diverse aspects of immune regulation. Here we show that 5-HT can efficiently drive programmed cell death in established Burkitt lymphoma (BL) lines that remain faithful to the original biopsy phenotype (group 1). Group 1 BL cells cultured in the presence of 5-HT exhibited marked suppression of DNA synthesis that was accompanied by extensive apoptosis—serotonin-driven apoptosis was complete within 24 hours, was preceded by early caspase activation, and was accompanied by a decline in mitochondrial membrane potential. BL cells that had drifted to a lymphoblastic group 3 phenotype were relatively resistant to these actions of serotonin, and the forced ectopic expression of either bcl-2 orbcl-xL provided substantial protection from 5-HT–induced apoptosis. 5-HT receptor antagonists (SDZ205-557, granisetron, methysergide) failed to inhibit serotonin-induced apoptosis, whereas the selective serotonin reuptake inhibitors (SSRI)—fluoxetine (Prozac), paroxetine (Paxil), and citalopram (Celexa)—substantially blocked the monoamine actions. Western blot analysis showed that BL cells expressed protein for the 5-HT transporter, and transport assays confirmed active uptake of serotonin by the cells. Unlike what was suggested for neuronal cells, there was no evidence that intracellular oxidative metabolites were responsible for the 5-HT–induced programmed death of BL cells. These data indicate that serotonin drives apoptosis in biopsylike BL cells after its entry through an active transport mechanism, and they suggest a novel therapeutic modality for Burkitt lymphoma.

SUMMARY We infected highly susceptible BALB/c and relatively resistant C57BL/6 mice with cloned Trypanosoma congolense and followed the effects of these infections on the circulating parasite numbers, mouse mortality and cytokine expression. C57BL/6 mice controlled their parasitaemia and survived for up to 1636 12 days, while BALB/c mice could not control their parasitaemia and succumbed to the infection within 8·46 0·5 days. Susceptible BALB/c mice had dramatically higher plasma levels of IL-10 than the resistant C57BL/6 mice from day 7 forward. This was preceded by an earlier and higher level induction of splenic IL-10 messenger RNA (mRNA) expression in the infected BALB/c mice. There was a strong negative correlation between the splenocyte proliferative responses to Concanavalin-A (Con-A) and their production of IL-10 in these infected BALB/c mice. Co-treatment of the Con-A-stimulated spleen cell cultures with monoclonal anti-IL-10 antibodies, but not isotypematched control antibodies, could completely reverse this suppression of the splenocyte proliferative response. Finally, in three experiments, anti-IL-10 antibody treatment in vivo reduced the peak circulating parasitaemia of infected BALB/c mice by 43% and increased their median survival periods by 38% relative to isotype-matched control antibody-treated mice.

Dendritic cells (DCs) are professional antigen-presenting cells of the immune system and can be generated in vitro from bone-marrow cells. In this study, we systematically investigated by DNA array analysis the expression profiles of 514 immunologically relevant genes in two populations of mouse bone marrow-derived DC, immature (DC(IMAT)), and lipopolysaccharide (LPS)-stimulated mature (DC(MAT)) DCs. Our data showed that DC(IMAT) expressed transcripts for 69 (13.42% of the 514) of these genes and that, upon maturation, 32 (6.23%) of these were up-regulated and 40 (7.78%) down-regulated. Maturation-dependent up-regulation, defined by a differential expression (DE) ratio of2, was observed among five cytokine (Flt-3L, TNF-alpha, IL-1alpha and -1beta, and IL-6), three chemokine (RANTES, MIP-2 and GROa) and three other (iNOS, MMP-13, and STRAP) genes. Reciprocally, maturation-dependent down-regulation occurred with one cytokine (IGF-1), two chemokine receptor (CCR2 and CCR5), and three other (RP105, Ax1, and UCP2) genes. Lower level, but nevertheless significantly enhanced expression of the chemokine receptor CCR7 and of NF-kappaB was also observed upon DC maturation. This DC maturation profile confirms previous findings from other lab, but it also substantially broadens our view of these cells by documenting expression changes among genes (e.g., IGF-1, MMP-13, STRAP) not reported previously in these cells.

Background Anaphylaxis is a life-threatening condition for which we have limited therapeutic options. Although specific immunotherapy for food allergies is becoming more effective, it is still laborious and carries substantial risk of adverse events. On the other hand, regulatory dendritic cell (DC) therapy is effective in mouse models of allergic disease and has been shown to work with TH2 cells from atopic asthmatic patients. Objective We assessed whether DC immunotherapy could reverse food allergen sensitivity in mouse models to provide proof of concept relating to their use in the clinic. Methods We generated and characterized mature retinoic acid-skewed dendritic cells (DC-RAs) and assessed their abilities to reverse ovalbumin or peanut allergies in mouse models, as well as their operative mechanisms. Results DC-RAs displayed a mature yet tolerogenic phenotype, expressing IL-10, TGF-β, IL-27, and aldehyde dehydrogenase 1A2 but not IL-12 or IL-35; IL-10 and TGF-β together drove their suppression of TH2 cell proliferation. Delivery of specific allergen-presenting DC-RAs to half-maximally sensitized mice with ovalbumin or peanut allergy reduced anaphylactic responses to oral allergen challenge by 84% to 90%, as well as diarrhea, mast cell activation, and TH2 cytokine responses and serum allergen-specific IgE/IgG1 levels. DC-RA expression of IL-27 was important to their induction of CD25+ lymphocyte activation gene 3 (LAG3)+, CD49b-, forkhead box P3 (Foxp3)- regulatory T cells in vitro, such that β subunit of IL-27 (Ebi)-/- (ie, IL-27-incompetent) DC-RAs were ineffective in inducing food allergen tolerance. Conclusion Our data indicate that regulatory DC immunotherapy can be effective for food allergies and suggest that induction of Foxp3- regulatory T cells might be a useful strategy for tolerance induction in this context.

Inhalation of agricultural occupational dusts from swine confinement facilities can result in lung inflammation. The innate immune response to organic barn dusts results in production of a number of pro-inflammatory factors in the lungs of barn workers such as cytokines, chemokines, and an influx of neutrophils. Many of these inflammatory factors are influenced by the chemokine CXCL8/IL-8 (KC or MIP-2 in mice). Previously, we have demonstrated that an endotoxin-independent component of swine barn dust extract (SBE) elevates lung chemokines in a protein kinase C (PKC)-dependent manner resulting in the significant formation of lung inflammatory cell infiltrates in a mouse model of SBE injury. In this study we test the ability of a CXCR1/CXCR2 antagonist, CXCL8(3-74)K11R/G31P (G31P) to block many of the features of lung-inflammation in response to challenge with SBE in an established mouse exposure system. Injection of G31P concurrent with SBE nasal instillation over a course of 3 weeks significantly reduced neutrophil accumulation in the lungs of barn dust exposed animals compared to those given SBE alone. There was a similar reduction in pro-inflammatory cytokines and chemokines IL-6, KC, and MIP-2 in SBE plus G31P-treated mice. In addition to excreted products, the receptors ICAM-1, CXCR1, and CXCR2, which all were elevated with SBE exposure, were also decreased with G31P treatment. SBE activation of PKCα and PKCe was reduced as well with G31P treatment. Thus, G31P was found to be highly effective at reducing several features of lung inflammation in mice exposed to barn dust extracts.

The need to activate B lymphocytes on antigen challenge is tempered by the requirement that the response is terminated when the challenge has been negotiated. Regulation is probably mediated via the interaction of soluble factors with surface receptors. Here, John Gordon and Graeme Guy review recent work that has focused on the actions of the soluble regulatory mediators and on the surface molecules they may interact with — emphasizing the growing importance of the CD23 antigen.

Cisplatin (DDP), a cytotoxic antitumor drug, functions in a dose-dependent manner. However, the pursuit for high‑dose therapeutic effects leads to more serious side effects including kidney toxicity. Nephrotoxicity caused due to endothelial cell dysfunction and neutrophils infiltration in kidneys. Interleukin-8 (IL-8) is an ELR+ chemokine binds with CXCR1/2 receptors and its role is primarily in neutrophils recruitment and also involved in invasion, angiogenesis and metastasis of different solid tumors including liver cancer. G31P, a CXCR1/2 antagonist, binds with CXCR1/2 with high affinity, and acts as an anti-inflammatory and antitumor agent. In the present study, we examined the antitumor effects of G31P and DDP on mouse liver cancer cells, and the effects exerted by G31P on cisplatin-induced renal injury. In vitro, effects of the G31P and DDP regimen on H22 cell proliferation were investigated by MTT assay. In vivo BALB/c mice were inoculated subcutaneously with 1x106 H22 cells and treated after one week with a high single dose of DDP with and without G31P on alternative days until the experiment was terminated. On the 15th day the mice were sacrificed, dissected and kidney tissues were analyzed using H&E staining. Myeloperoxidase (MPO) activity was assessed and RT-PCR was performed to detect inflammatory cytokines. Solid tumors were weighed for tumor growth and performed pathological examination, immunohistochemistry and western blotting were performed to detect tissue-related protein expressions in tumor tissue. The tumor inhibitory rate of DDP, G31P and DDP+G31P groups was 38.40, 40.74 and 74.80%, respectively, and the general state of mice in the DDP+G31P group was significantly improved as compared to the DDP group. The results indicated that G31P with DDP significantly inhibited the proliferation while the growth of H22 cell carnimona in vitro and in vivo enhanced the efficacy of cisplatin in cancer treatment with reduced side effects on acute renal failure.

Background Proteinase-activated receptor 2 (PAR2 ) is a G protein-coupled receptor activated by trypsin-like serine proteinases. PAR2 activation has been associated with inflammation including allergic airway inflammation. We have also shown that PAR2 activation in the airways leads to allergic sensitization. The exact contribution of PAR2 in the development of eosinophilic inflammation and airway hyperresponsiveness (AHR) in sensitized individuals is not clear. Objective To investigate whether functional inhibition of PAR2 during allergen challenge of allergic mice would inhibit allergen-induced AHR and inflammation in mouse models of asthma. Methods Mice were sensitized and challenged with ovalbumin (OVA) or cockroach extract (CE). To investigate the role of PAR2 in the development of AHR and airway inflammation, we administered blocking anti-PAR2 antibodies, or a cell permeable peptide inhibitor of PAR2 signalling, pepducin, i.n. before allergen challenges and then assessed AHR and airway inflammation. Results Administration of anti-PAR2 antibodies significantly inhibited OVA- and CE-induced AHR and airway inflammation. In particular, two anti-PAR2 antibodies, the monoclonal SAM-11 and polyclonal B5, inhibited AHR, airway eosinophilia, the increase of cytokines in the lung tissue and antigen-specific T cell proliferation, but had no effect on antigen-specific IgG and IgE levels. Pepducin was also effective in inhibiting AHR and airway inflammation in an OVA model of allergic airway inflammation. Conclusions and clinical relevance Functional blockade of PAR2 in the airways during allergen challenge improves allergen-induced AHR and inflammation in mice. Therefore, topical PAR2 blockade in the airways, through anti-PAR2 antibodies or molecules that interrupt PAR2 signalling, has the potential to be used as a therapeutic option in allergic asthma.

We systematically investigated the impact of the relative maturation levels of dendritic cells (DCs) on their cell surface phenotype, expression of cytokines and chemokines/chemokine receptors (by DNA array and RNase protection analyses), biological activities, and abilities to induce tumor immunity. Mature DCs expressed significantly heightened levels of their antigen-presenting machinery (e.g., CD54, CD80, CD86) and numerous cytokines and chemokines/chemokine receptors (i.e., Flt-3L, G-CSF, IL-1alpha and -1beta, IL-6, IL-12, CCL-2, -3, -4, -5, -17, and -22, MIP-2, and CCR7) and were significantly better at inducing effector T cell responses in vitro. Furthermore, mice vaccinated with tumor peptide-pulsed mature DCs better survived challenge with a weakly immunogenic tumor (8 of 8 survivors) than did mice vaccinated with less mature (3 of 8 survived) or immature (0 of 8 survivors) DCs. Nevertheless, intermediate-maturity DCs expressed substantial levels of Flt-3L, IGF-1, IL-1alpha and -1beta, IL-6, CCL-2, -3, -4, -9/10, -17, and -22, MIP-2, osteopontin, CCR-1, -2, -5, and -7, and CXCR-4. Taken together, our data clearly underscore the critical nature of employing DCs of full maturity for DC-based antitumor vaccination strategies.

OBJECTIVE To map the expression of transforming growth factor (TGF)-beta(1), TGF-beta(3), and basic fibroblast growth factor (bFGF) in full-thickness skin wounds of the horse. To determine whether their expression differs between limbs and thorax, to understand the pathogenesis of exuberant granulation tissue. STUDY DESIGN Six wounds were created on one lateral metacarpal area and one midthoracic area of each horse. Sequential wound biopsies allowed comparison of the temporal expression of growth factors between limb and thoracic wounds. ANIMALS Four 2- to 4-year-old horses. METHODS Wounds were assessed grossly and histologically at 12 and 24 hours, and 2, 5, 10, and 14 days postoperatively. ELISAs were used to measure the growth factor concentrations of homogenates of wound biopsies taken at the same timepoints. RESULTS TGF-beta(1) peaked at 24 hours in both locations and returned to baseline in thoracic wounds by 14 days but remained elevated in limb wounds for the duration of the study. Expression kinetics of TGF-beta(3) differed from those of TGF-beta(1). TGF-beta(3) concentrations gradually increased over time, showing a trend toward an earlier and higher peak in thoracic compared with limb wounds. bFGF expression kinetics resembled those of TGF-beta(1), but no statistically significant differences existed between limb and thoracic wounds. CONCLUSIONS Growth factor expression is up-regulated during normal equine wound repair. TGF-beta(1) and TGF-beta(3) show a reciprocal temporal regulation. Statistically significant differences exist between limb and thoracic wounds with respect to TGF-beta(1) expression. CLINICAL RELEVANCE The persistence of TGF-beta(1) expression in leg wounds may be related to the development of exuberant granulation tissue in this location, because TGF-beta(1) is profibrotic.

Gaspar, K. J., K. J. Racette, J. R. Gordon, M. E. Loewen, and G. W. Forsyth. Cloning a chloride conductance mediator from the apical membrane of porcine ileal enterocytes. Physiol Genomics 3: 101–111, 2000.—Attempts to attribute ileal brush-border chloride conductance to specific proteins were pursued by screening a porcine intestinal cDNA library. A 0.94-kb clone was identified on expression screening with a monoclonal antibody that inhibited enterocyte brush-border chloride conductance. Further screening approaches led to the isolation of a 3.1-kb full-length sequence called pCLCA1, consistent with the identification of a 2.9-kb transcript through Northern analysis. This sequence had significant homology to the CLCA gene family of calcium-regulated chloride channels, especially to hCLCA1. However, a strong A-kinase consensus phosphorylation site in a predicted cytoplasmic loop of the protein was a notable difference from the hCLCA1 gene product. Several porcine exocrine epithelial tissues, including ileum, trachea, and the major salivary glands express pCLCA1 mRNA. In situ hybridization studies localized the expression of pCLCA1 mRNA to the crypt and villus epithelia of porcine ileum, whereas tracheal expression was observed in both surface epithelium and submucosal glands. In situ expression of pCLCA1 in mouse 3T3 cells induces an ionomycin-dependent chloride conductance activity in these cells.

Resistance to African trypanosomiasis is under multigenic control. BALB/c mice are highly susceptible while C57Bl/6 mice are relatively resistant. Macrophages eliminate opsonized trypanosomes from the bloodstream and are involved in immunosuppression. We therefore investigated the production of a number of cytokines (IL-10, IL-6, TNF-alpha and IL-12) by bone marrow-derived macrophages (BMDM) from C57Bl/6 and BALB/c mice following challenge with either Trypanosoma congolense or Trypanosoma brucei. BMDM from C57Bl/6 mice, upon challenge with whole cell extracts (WCE) of T. congolense or T. brucei, produced significantly more TNF-alpha and IL-12 than those from BALB/c mice. The production of these cytokines was significantly enhanced by pretreatment of the cells with IFN-gamma. BMDM from BALB/c mice, however, produced significantly more IL-6 and IL-10 than those from C57Bl/6 mice. In contrast to LPS stimulation, simultaneous treatment of cells with WCE and IFN-gamma enhanced IL-10 synthesis by BMDM from BALB/c mice. These results indicate that cytokine genes are differentially regulated in macrophages from trypanosome-susceptible and -resistant mice and are consistent with our previous findings wherein retrovirus-immortalized macrophage cell lines from BALB/c and C57Bl/6 mice produce differential amounts of cytokines after phagocytosis of trypanosomes.

Background: Macrophages and infiltrating monocytes comprise the largest population of cells within the airways of patients with allergic asthma. Both become activated and thus contribute to the pathologic features of allergic reactions, but the mechanism by which they are recruited has not been well documented. Objective: Our purpose was to assess the role of the CC chemokine monocyte chemotactic peptide-1 (MCP-1) in monocyte recruitment during allergic reactions in mice. Method: We used immunohistochemistry and Northern blotting to assess MCP-1 expression, selective mast cell reconstitution of mast cell–deficient W/W v mice to demonstrate the mast cell dependence of MCP-1 expression and monocyte recruitment and neutralizing anti-MCP-1 antibodies to block monocyte recruitment during cutaneous allergic reactions. Results: MCP-1 was expressed largely by resident dermal cells within the allergic lesions at 4 hours after challenge, followed within several hours by an influx of monocytes; at 10 hours after challenge monocytes comprised a substantial proportion of the infiltrating cells at the nidus of the response. Mast cell–reconstituted, but not mast cell–deficient W/W v mice expressed MCP-1 transcripts and developed monocyte infiltrates after allergen challenge. Finally, anti-MCP-1 antibody treatments reduced by ~63% the influx of monocytes into the reaction sites. Conclusions: These data clearly demonstrate the mast cell dependence of the MCP-1 expression and the monocyte influx and establish a substantial, but not exclusive, causal relationship between these 2 events. (J Allergy Clin Immunol 2000;106:110-6.)

Monocytes/macrophages usually make up the largest population of cells in the airways of allergic asthma patients and, as such, contribute substantially to the pathogenesis of this and other allergic diseases. In this report we address one mechanism by which monocytes can be recruited during allergic responses. We and others have shown previously that MCP-1 is important to monocyte infiltration of the tissues during allergic responses in mice and, independently, that mast cells activate fibroblasts to express type alpha1(I)-collagen during such responses. We demonstrate herein that immunologically activated, but not quiescent mouse bone-marrow-derived mast cells release mediators which in turn activate primary cultures of embryonic dermal fibroblasts for high-level secretion of monocyte chemoattractant activities. We identify the CC chemokine MCP-1 as a major component of this activity. Anti-MCP-1 antibodies neutralized approximately 80% of the monocyte chemoattractant activities secreted by such mast-cell-activated fibroblasts. Furthermore, our data implicate mast cell TGFbeta and TNFalpha in this process. Depletion of TGFbeta, TNFalpha, or both TGFbeta and TNFalpha from the mediator pool secreted by mast cells activated via the FcepsilonRI reduced the mast-cell-driven fibroblast MCP-1 response by 80+/-15, 56+/-11, or 82+/-5%, respectively. These data thus further delineate a mechanism by which fibroblasts are recruited into and participate in the mast cell-leukocyte cytokine cascades that orchestrate allergic responses.

BALB/c and C57B1/6 mice differ in resistance to Trypanosoma congolense infections. Evidence suggests that macrophages play a central role in the resistance to trypanosomiasis. Nitric oxide (NO) produced by macrophages in response to various stimuli or pathogens is one of the important arms of nonspecific immunity. We investigated the production of NO by the peritoneal macrophages and bone marrow-derived macrophages (BMDM) from trypanosome-resistant C57B1/6 and -susceptible BALB/c mice following stimulation with T. congolense whole cell extract (WCE) or following phagocytosis of T. congolense mediated by anti-variant surface glycoprotein (VSG) antibodies of IgM or IgG2a isotype. C57B1/6 peritoneal macrophages as well as BMDM produced significantly more NO than similar BALB/c macrophages in response to T. congolense lysate and IFN-gamma. In both BALB/c and C57B1/6 BMDM cultures, phagocytosis of T. congolense mediated by anti-VSG antibodies of IgG2a isotype in the presence of IFNgamma induced two- to ninefold more NO than phagocytosis mediated by IgM antibodies and C57B1/6 BMDM produced significantly higher amounts of NO than BALB/c BMDM under these conditions. NO produced by BMDM was found to exert trypanostatic effect on T. congolense in vitro, but was not found to influence the in vivo infectivity of these treated parasites under the conditions used in this study.

BALB/c and C57Bl/6 mice differ in resistance to T. congolense infections. We investigated the production of various cytokines (IL-10, IL-6, TNF-alpha and TGF-beta) by macrophages from these mice. Macrophage cell lines (BALB.BM cells) of BALB/c mice but not (ANA-I cells)of C57BL/6 mice constitutively produced IL-10. Challenge of these cells with trypanosomes induced the production of 50-100 times more IL-10 in BALB.BM cells than in ANA-1 cells. Pre-incubation of the cell lines with IFN-gamma. prior to the trypanosome challenge, further upregulated this IL-10 production in BALB.BM but not in ANA-1 cells. Primary cultures of bone marrow-derived macrophages (BMDM) from BALB/c mice also produced more IL-10 following challenge with IFN-gamma and opsonized trypanosomes than did the C57Bl/6 BMDM. Similarly after challenge with trypanosomes, BALB.BM and BALB/c BMDM produced significantly more IL-6 than did the analogous cells from the C57Bl/6 mice following such challenges. Higher steady state levels of TNF-alpha mRNA accumulated in ANA-1 cells than in BALB.BM cells following challenge with IFN-gamma and opsonized trypanosomes. Findings of this study for the first time indicate a differential regulation of cytokines (IL-10, IL-6 and TNF-alpha) in macrophages of mice that significantly differ in their susceptibility to infections with T. congolense.