Project description:Activated allergen-specific Th2 and Th1 cells release cytokines that transform neutrophils into functional APCs characterized by the expression of HLA-DR and CD58 as well as enhanced survival and antigen uptake, irrespectively of the presence of IL-10, which reduces allergen uptake by neutrophils.
Project description:Dendritic cells (DC) are the professional antigen presenting cells (APC) that bridge the innate and adaptive immune system. Previously, in a CD11c/EYFP transgenic mouse developed to study DC functions, we anatomically mapped and phenotypically characterized a discrete population of EYFP(+) cells within the microglia that we termed brain dendritic cells (bDC). In this study, we advanced our knowledge of the function of these cells in the CD11c/EYFP transgenic mouse and its chimeras, using acute stimuli of stereotaxically inoculated IFNgamma or IL-4 into the CNS. The administration of IFNgamma increased the number of EYFP(+)bDC but did not recruit peripheral DC into the CNS. IFNgamma, but not IL-4, upregulated the expression levels of major histocompatibility class II (MHC-II). In addition, IFNgamma-activated EYFP(+)bDC induced antigen-specific naïve CD4 T cells to proliferate and secrete Th1/Th17 cytokines. Activated bDC were also able to stimulate naïve CD8 T cells. Collectively, these data reveal the Th1 cytokine IFNgamma, but not the Th2 cytokine IL4, induces bDC to up-regulate MHC-II and become competent APC.
Project description:Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.
Project description:The high affinity IgE receptor, Fc epsilonRI, plays a key role in the immunological pathways involved in allergic asthma. Previously we have demonstrated that human neutrophils isolated from allergic asthmatics express a functional Fc epsilonRI, and therefore it was of importance to examine the factors regulating its expression. In this study, we found that neutrophils from allergic asthmatics showed increased expression of Fc epsilonRI-alpha chain surface protein, total protein and mRNA compared with those from allergic non asthmatics and healthy donors (p<0.001). Interestingly, in neutrophils isolated from allergic asthmatics, Fc epsilonRI-alpha chain surface protein and mRNA expression were significantly greater during the pollen season than outside the pollen season (n = 9, P = 0.001), an effect which was not observed either in the allergic non asthmatic group or the healthy donors (p>0.05). Allergen exposure did not affect other surface markers of neutrophils such as CD16/Fc gammaRIII or IL-17R. In contrast to stimulation with IgE, neutrophils incubated with TH2 cytokines IL-9, GM-CSF, and IL-4, showed enhanced Fc epsilonRI-alpha chain surface expression. In conclusion, these results suggest that enhanced Fc epsilonRI expression in human neutrophils from allergic asthmatics during the pollen season can make them more susceptible to the biological effects of IgE, providing a possible new mechanism by which neutrophils contribute to allergic asthma.
Project description:The phagocytosis-promoting complement receptor, Complement Receptor Immunoglobulin (CRIg), is exclusively expressed on macrophages. It has been demonstrated that expression in macrophages could be modulated by inflammatory mediators, including cytokines. This raised the possibility that a major phagocyte, the neutrophil, may also express CRIg following activation with inflammatory mediators. Here we show that resting peripheral blood neutrophil lysates subjected to protein analysis by Western blot revealed a 35 kDa CRIg isoform, consistent with the expression of CRIg mRNA by RT-PCR. By flow cytometry, CRIg was detected intracellularly and in very minor amounts on the cell surface. Interestingly, expression on the cell surface was significantly increased to functional levels after activation with inflammatory mediators/neutrophil activators; N-Formylmethionine-leucyl-phenylalanine, tumor necrosis factor (TNF), Granulocyte-Macrophage Colony stimulating Factor (GM-CSF), bacterial lipopolysaccharide, leukotriene B4 and phorbol myristate acetate. The increase in expression required p38 MAP kinase and protein kinase C activation, as well as intracellular calcium. Neutrophils which were defective in actin microfilament reorganization due to a mutation in ARPC1B or inhibition of its upstream regulator, Rac2 lose their ability to upregulate CRIg expression. Inhibition of another small GTPase, Rab27a, with pharmacological inhibitors prevented the increase in CRIg expression, suggesting a requirement for the actin cytoskeleton and exocytosis. Engagement of CRIg on TNF-primed neutrophils with an anti-CRIg monoclonal antibody increased the release of superoxide and promoted the activation of p38 but not ERK1/ERK2 or JNK MAP kinases. The TNF-induced increase in killing of Staphylococcus aureus was blocked by the anti-CRIg antibody. Adding to the anti-microbial role of CRIg, it was found that GM-CSF priming lead to the release of neutrophil extracellular traps. Interestingly in contrast to the above mediators the anti-inflammatory cytokine IL-10 caused a decrease in basal expression and GM-CSF induced increase in CRIg expression. The data demonstrate that neutrophils also express CRIg which is regulated by inflammatory mediators and cytokines. The findings show that the neutrophil antimicrobial function involving CRIg requires priming as a means of arming the cell strategically with microbial invasion of tissues and the bloodstream.
Project description:Neutrophils are particularly well known for their antimicrobial function. Although historically they are regarded as strictly a phagocyte of the innate immune system, over time it has become clear that neutrophils are versatile cells with numerous functions including innate and adaptive immune regulation. We have previously described a role for human neutrophils in antibody-mediated red blood cell (RBC) clearance. Under homeostatic conditions, neutrophils do not take up RBCs. Yet, when RBCs are immunoglobulin G (IgG) opsonized, which can occur in alloimmunization or autoimmunization reactions, neutrophils can effectively phagocytose RBCs. In the present study, we show that human neutrophils acquire an antigen-presenting cell (APC) phenotype following RBC phagocytosis. Subsequent to RBC phagocytosis, neutrophils expressed major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40 and CD80. Moreover, in classical APCs, the respiratory burst is known to regulate antigen presentation. We found that the respiratory burst in neutrophils is reduced after IgG-mediated RBC phagocytosis. Additionally, following RBC phagocytosis, neutrophils were demonstrated to elicit an antigen-specific T-cell response, using tetanus toxoid (TT) as an antigen to elicit an autologous TT-specific CD4+ T-cell response. Lastly, although the "don't eat me" signal CD47 is known to have a powerful restrictive role in the activation of immunity against RBCs in dendritic cells, CD47 does not seem to have a significant effect on the antigen-presenting function of neutrophils in this context. Overall, these findings reveal that besides their classical antimicrobial role, neutrophils show plasticity in their phenotype.
Project description:BackgroundLittle is known about the effects of socioeconomic status or cockroach allergen on immune responses in school-age children, particularly in tropical environments.ObjectiveTo examine whether cockroach allergen and/or socioeconomic status is associated with plasma cytokine levels in Puerto Rican children.MethodsThis was a cross-sectional study of 532 children (6-14 years old) with (n = 272) and without (n = 260) asthma in San Juan (Puerto Rico). House dust allergens (cockroach [Bla g 2], dust mite [Der p 1], cat dander [Fel d 1], dog dander [Can f 1], and mouse urinary protein [Mus m 1]) were quantified using monoclonal antibody arrays. A panel of 14 cytokines (interleukin [IL]-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interferon-γ, and tumor necrosis factor-α) was measured in plasma samples. Low household income was defined as less than $15,000 per year (below the median income for Puerto Rico in 2008-2009). Linear regression was used for the analysis of cockroach allergen and plasma cytokines.ResultsIn a multivariable analysis adjusting for low income and other allergen levels, cockroach allergen was significantly associated with decreased IL-17A and with increased levels of 8 cytokines (IL-4, IL-10, IL-17F, IL-21, IL-25, IL-31, interferon-γ, and tumor necrosis factor-α). After stratifying this analysis by cockroach allergy (ie, having a cockroach positive immunoglobulin E reaction), our findings remained largely unchanged for children sensitized to cockroach but became weaker and statistically nonsignificant for non-sensitized children.ConclusionCockroach allergen has broad effects on adaptive immune responses in school-age children in a tropical environment, particularly in those sensitized to cockroach.
Project description:Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow-derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture-induced sepsis but not in CIRP-/- mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non-APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4+ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4+ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4+ T cell activation, Th1 polarization, and IFN-γ-mediated hyper-NETosis.
Project description:Active episodes of the inflammatory bowel diseases are associated with the infiltration of large numbers of myeloid cells including neutrophils, monocytes, and macrophages. The objective of this study was to systematically characterize and define the different populations of myeloid cells generated in a mouse model of chronic gut inflammation. Using the T cell transfer model of chronic colitis, we found that induction of disease was associated with enhanced production of myelopoietic cytokines (IL-17 and G-CSF), increased production of neutrophils and monocytes, and infiltration of large numbers of myeloid cells into the mesenteric lymph nodes (MLNs) and colon. Detailed characterization of these myeloid cells revealed three major populations including Mac-1(+)Ly6C(high)Gr-1(low/neg) cells (monocytes), Mac-1(+)Ly6C(int)Gr-1(+) cells (neutrophils), and Mac-1(+)Ly6C(low/neg)Gr-1(low/neg) leukocytes (macrophages, dendritic cells, and eosinophils). In addition, we observed enhanced surface expression of MHC class II and CD86 on neutrophils isolated from the inflamed colon when compared with neutrophils obtained from the blood, the MLNs, and the spleen of colitic mice. Furthermore, we found that colonic neutrophils had acquired APC function that enabled these granulocytes to induce proliferation of OVA-specific CD4(+) T cells in an Ag- and MHC class II-dependent manner. Finally, we observed a synergistic increase in proinflammatory cytokine and chemokine production following coculture of T cells with neutrophils in vitro. Taken together, our data suggest that extravasated neutrophils acquire APC function within the inflamed bowel where they may perpetuate chronic gut inflammation by inducing T cell activation and proliferation as well as by enhancing production of proinflammatory mediators.
Project description:Cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-8 attract neutrophils into inflammatory sites. During emigration from the blood neutrophils interact with extracellular matrix proteins such as fibronectin. Fibronectin provides beta2-integrin co-stimulation, allowing GM-CSF and IL-8 to activate nuclear factor (NF)-kappaB, an effect that does not occur in suspension. We tested the hypothesis that exposure of mice to fever-like temperatures abrogates neutrophil recruitment and NF-kappaB activation in a mouse model of skin inflammation. Mice that were exposed to 40 degrees C for 1 hour showed strongly reduced GM-CSF- and IL-8-induced neutrophilic skin inflammation. In vitro heat exposure did not interfere with neutrophil adhesion or spreading on fibronectin but strongly inhibited migration toward both cytokines. Using specific inhibitors, we found that PI3-K/Akt was pivotal for neutrophil migration and that heat down-regulated this pathway. Furthermore, neutrophils on fibronectin showed abrogated NF-kappaB activation in response to GM-CSF and IL-8 after heat. In vivo heat exposure of mice followed by ex vivo stimulation of isolated bone marrow neutrophils confirmed these results. Finally, less NF-kappaB activation was seen in the inflammatory lesions of mice exposed to fever-like temperatures as demonstrated by in situ hybridization for IkappaBalpha mRNA. These new findings suggest that heat may have anti-inflammatory effects in neutrophil-dependent inflammation.