Sensitization to fungi often leads to a severe form of asthma that is particularly difficult to manage clinically, resulting in increased morbidity and hospitalizations in these patients. Although B lymphocytes might exacerbate asthma symptoms through the production of IgE, these cells might also be important in the protective response against inhaled fungi. Through cytokine release and T-cell interactions, these lymphocytes might also influence the development and maintenance of airway wall fibrosis. J H 2/2 mice lack the JH gene for the heavy chain component of antibodies, which is critical for B-cell function and survival. These animals have facilitated the elucidation of the role of B lymphocytes in a number of immune responses; however, J H 2/2 mice have not been used to study fungal allergy. In this study, we examined the role of B lymphocytes using an Aspergillus fumigatus murine fungal aeroallergen model that mimics human airway disease that is triggered by environmental fungal exposure. We compared disease progression in sensitized wild-type BALB/c and J H 2/2 mice that were exposed to repeated fungal exposure and found no differences in airway hyperresponsiveness, overall pulmonary inflammation or collagen deposition around the large airways. However, the levels of the Th2-type cytokines IL-4 and IL-13 were significantly attenuated in the airways of J H 2/2 mice relative to the BALB/c controls. By contrast, levels of the inflammatory cytokines IL-17A and IL-6 were significantly elevated in the J H 2/2 animals, and there was significantly more robust airway eosinophilia and neutrophilia than in control animals. Taken together, these findings demonstrate that B lymphocytes help to regulate granulocytic responses to fungal exposure in the pulmonary compartment.
Allergic asthma is a chronic inflammatory disease of the airways characterized by excessive eosinophilic and lymphocytic inflammation with associated changes in the extracellular matrix (ECM) resulting in airway wall remodeling. Hyaluronan (HA) is a nonsulfated glycosaminoglycan ECM component that functions as a structural cushion in its high molecular mass (HMM) but has been implicated in metastasis and other disease processes when it is degraded to smaller fragments. However, relatively little is known about the role HA in mediating inflammatory responses in allergy and asthma. In the present study, we used a murine Aspergillus fumigatus inhalational model to mimic human disease. After observing in vivo that a robust B cell recruitment followed a massive eosinophilic egress to the lumen of the allergic lung and corresponded with the detection of low molecular mass HA (LMM HA), we examined the effect of HA on B cell chemotaxis and cytokine production in the ex vivo studies. We found that LMM HA functioned through a CD44-mediated mechanism to elicit chemotaxis of B lymphocytes, while high molecular mass HA (HMM HA) had little effect. LMM HA, but not HMM HA, also elicited the production of IL-10 and TGF-β1 in these cells. Taken together, these findings demonstrate a critical role for ECM components in mediating leukocyte migration and function which are critical to the maintenance of allergic inflammatory responses.
Vasoactive intestinal peptide receptor – 1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.
Vasoactive intestinal peptide (VIP) is a potent chemoattractant for immune cells that is delivered by the peripheral nervous system to immune organs. However, the molecular mechanism regulating VIP’s chemoattractant properties is not known. Previously, our group published the VIP transcriptome in resting CD4 T cells, which identified a chemoattractant gene set (e.g. EGFR, Snail and syt XIII) that was coordinately upregulated. We hypothesize that these gene products control the chemoattractant activity of VIP in immune cells. Moreover, we propose that this chemoattractant gene set is transcriptionally upregulated by the transactivation of EGFR by VIP as is observed in non‐immune cells, including breast and colon cancer cells. This research will utilize primary mouse splenocytes treated +/‐ VIP over various time intervals in an attempt to identify which FACS‐sorted splenocyte subpopulations are sensitive to VIP and the extent to which the chemoattractant gene set is upregulated. Optimal conditions will be repeated +/‐ EGFR neutralizing antibody or a specific EGFR kinase inhibitor to assess the contribution of EGFR enzymatic activity for the chemoattractant gene expression changes. Lastly, Boyden‐chamber chemotaxis assays will be employed to test whether pharmacological inhibition against EGFR kinase activity and/or other chemoattractant gene products suppresses VIP’s chemoattractant activity using identified splenocyte subpopulations. The significance of the proposed research is to better understand the molecular control of lymphocyte trafficking mediated by the nervous system. Grant Funding Source: This research was supported by NIH/NIAID (R15 to GD).
Effective immune protection against enteric pathogens is critical for the survival of the mammalian host. The human gut is home to nearly 100 trillion microorganisms consisting of ≈1000 distinct bacterial species, some of which are life‐threatening if they escape the gut lumen. Recruitment of peripheral, naïve T Lymphocytes to the gut replenishes this organ with regulatory T cells and folicular helper T cells, as well as assisting in B cell activation within specilizaed gut‐lymphoid regions, called Peyer's Patches. A potent T cell chemoattractant, called vasoactive intestinal peptide (VIP), is secreted by a dense network of nerves innervating the gut. Importantly, the removal of VIP receptors expressed on T cells causes a drastic reduction in recruited gut T cells supporting a role for this signaling axis in T cell trafficking to this organ. However, the molecular mechanism controlling T cell homing to the gut by VIP is presently unknown. We hypothesized that VIP signaling upregulates an epidermal growth factor receptor (EGFR) signaling pathway, based on a previously published global microarray study by our laboratory. Using primary, resting mouse CD4 T cells, we tested whether a coordinated upregulation of this EGFR pathway occurred by qRT‐PCR. This analysis showed a rapid, transient and coordinated upregulation of the EGFR pathway ≤ 2.5 hours post‐exogenous VIP treatment. Collectively, we conclude that VIP signaling initiates a chemoattractant cellular program through the coordinated upregulation of the EGFR pathway. Future research will be required to directly test whether this mechanism controls resting T cell trafficking to the gut.
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