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IgG Fc Receptor Activity in vivo is Under Complement Control
Abstracts AB217
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2
Interleukin- 4 Induces Bronchial Epithelial Cells Barrier Dysfunction B. Saatian, N. Meednu, F. Rezaee, L. Beck, S. N. Georas; University of Rochester Medical Center, Rochester, NY. RATIONALE: Airway epithelia actively participate in lung defense against respiratory pathogens and allergens. The integrity of airway epithelial cells is essential for the maintenance of barrier function and paracellular permeability. Emerging evidence indicates that epithelial barrier function is compromised in asthma, a Th2-dominant disease, but the mechanisms involved are not well understood. The purpose of this study was to investigate the role of Th2-type cytokines on airway epithelial barrier function. METHODS: 16HBE14o- human bronchial epithelial cells monolayers were grown on collagen coated Transwell inserts. The basolateral or apical surfaces of airway epithelia were exposed to human interleukin-4 (hIL-4), TSLP, IL-25, or IL-33 alone or in combination at various concentrations and time points. We analyzed epithelial apical junctional complex (AJC) function by measuring transepithelial electrical resistance (TEER) and permeability to sodium fluorescein (NaF) over time. RESULTS: Transepithelial resistance was significantly decreased after basolateral (but not apical) exposure to IL-4 at 48 and 72 hours, with optimal IL-4 concentrations between 5-50 ng/ml. IL-4 also induced increased epithelial permeability as shown by increased apical- to basal flux of NaF. None of the other cytokines examined significantly affected TEER. Wortmannin, an inhibitor of PI3 kinase signaling pathway, did not block Il-4 mediated TEER reduction. CONCLUSIONS: Our study indicates that IL-4 has a disruptive effect on airway epithelial barrier function. IL-4 induced epithelial barrier dysfunction may contribute to airway inflammation in allergic asthma. The molecular mechanisms by which IL-4 increased bronchial epithelial cell permeability require further investigation.
817
Mutants of the Major Cockroach Allergen, Bla g 2, Modulate T Cell Responses in Cockroach-Allergic Subjects P. W. Wright1, J. Glesner2, M. Chapman2, A. Pomes2, J. Woodfolk1; 1 University of Virginia, Charlottesville, VA, 2Indoor Biotechnologies Inc., Charlottesville, VA. RATIONALE: The majority of cockroach (CR) allergic subjects have IgE antibodies to Bla g 2. Our objective was to test whether variants of Bla g 2 that contain mutations in conformational epitopes have the potential to modulate T cell responses and provide candidates for immunotherapy. METHODS: PBMCs from CR-allergic subjects (6 anti-Bla g 2 IgE antibodies) were stimulated with 12 Bla g 2 mutants (containing up to 3 point mutations), most of which showed reduced monoclonal antibody and IgE antibody binding. Cytokine responses were assessed by intracellular staining with flow cytometry, and by analysis of culture supernatants by cytometric bead assay. T cell proliferation was assessed by [3H] thymidine incorporation. RESULTS: Both wildtype Bla g 2 and mutants induced IFN-g+CD4+ T cells, with few IL-4+ or IL-10+ cells, regardless of sensitivity to Bla g 2. In cultures from Bla g 2-sensitized subjects, the deglycosylated variant N268Q enhanced secretion of IFN-g, IL-5, and IL-13. This mutant also increased TNF-a secretion, and the proportion of IFN-g+ T cells co-expressing TNF-a. These effects were generally diminished when additional mutations were introduced. In contrast to other cytokines, the variable profile of IL-10 secretion induced by different mutants (likely APC-derived) was remarkably consistent among subjects. Mutants inducing the highest proliferation contained substitutions that mapped to discrete molecular regions, but did not always induce the highest cytokine release, indicating the capacity to uncouple T-cell proliferation and cytokine pathways. CONCLUSIONS: Bla g 2 mutants modulate a Th1-dominated response and have the potential to enhance immunogenicity with or without inducing pro-inflammatory cytokines.
IgG Fc Receptor Activity in vivo is Under Complement Control E. Y. Wu, H. Jiang, G. Hester, M. M. Frank; Duke University Medical Center, Durham, NC. RATIONALE: Immunoglobulin G (IgG) receptors (FcgR) possess a critical role in the pathogenesis of immune-complex (IC) diseases. Previous reports suggest complement does not affect IC interaction with FcgR, but we assert the classical complement pathway down-regulates IC binding to FcgR. The Arthus reaction is a model of IC-mediated vasculitis, and we performed reverse passive Arthus reactions (RPAR) in normal and C1q-deficient (C1q-/-) C57BL/6 mice. We hypothesized the absence of C1q would enhance IC-mediated tissue damage. METHODS: Sedated and shaved mice (9 normal, 10 C1q-/-) were injected intradermally with 20 ml containing PBS alone on one side and affinity purified rabbit anti-BSA IgG 5 mg on the opposing side. Immediately after, purified BSA 100 mg and 125Iodine-labeled BSA 1.25 mg in PBS containing 1% Evans blue was injected intravenously. After 4 hours, skin sections were weighed and radioactivity was measured in cpm/gm of tissue. An Arthus index (AI) for each mouse was calculated by dividing cpm/gm of treated skin by cpm/gm of control skin. The Mann-Whitney U test was used to evaluate for significant difference between the median AI of the two mice groups. RESULTS: C1q-/- mice exhibited more vigorous RPAR (mean AI 3.660.8) than normal mice (mean AI 2.561.3). The median AI for C1q-/- mice was significantly higher than normal mice (3.8 vs 2.3, two-tail P<0.05). CONCLUSIONS: Our results provide physiologic evidence that the classical complement pathway down-regulates in vivo IC and FcgR mediated inflammation. They may also further understanding of why individuals with classical pathway defects are susceptible to developing autoimmune disease.
818
819
Effects of Vitamin D Deficiency on Inner City Children and Adolescents with HIV P. Poowuttikul, E. Secord; Children’s Hospital of Michigan, Wayne State University, Detroit, MI. RATIONALE: We previously reported high incidence of vitamin D deficiency in our HIV patients (primarily African American and living in low sunlight environment). We sought to identify any correlation with HIV disease markers and medications. METHODS: Serum 25 hydroxyvitamin D (25 OHD) was obtained from 160 HIV infected youth (25 OHD < 20 ng/mL classified as vitamin D deficiency; 20-35 ng/mL classified as vitamin D insufficiency). 25 OHD levels were correlated to HIV RNA (VL) and CD4+ T-cells. RESULTS: 152/160 (95%) youth had 25 OHD insufficiency. Out of these, 110/160 (68.8%) had deficiency. 24/110 (21.8%) of 25 OHD deficiency patients had undetectable VL as opposed to 15/50 (30%) with 25 OHD > 20 (p-value 5 0.15). CD4+ T-cell count was not significantly different between youth with insufficiency vs deficiency. Treatment with tenofovir or efavirenz significantly increased probability of vitamin D deficiency (48/66; 72.7% vs 27/49; 55.1%; p-value 5 0.074). We did not have sufficient youth with HIV and normal vitamin D levels to make any comparisons. CONCLUSIONS: Very high prevalence of vitamin D deficiency was confirmed. Higher 25 OHD tended to predict higher viral loads. No effect on CD4 + T cell count was observed. Treatment with tenofovir or efavirenz significantly increased the chance of vitamin D deficiency, as previously reported. HIV infection in African American youth in low sunlight areas are at significant risk for low vitamin D. Vitamin D supplementation should be routine in this population.
TUESDAY
816
J ALLERGY CLIN IMMUNOL VOLUME 129, NUMBER 2
Interleukin- 4 Induces Bronchial Epithelial Cells Barrier Dysfunction B. Saatian, N. Meednu, F. Rezaee, L. Beck, S. N. Georas; University of Rochester Medical Center, Rochester, NY. RATIONALE: Airway epithelia actively participate in lung defense against respiratory pathogens and allergens. The integrity of airway epithelial cells is essential for the maintenance of barrier function and paracellular permeability. Emerging evidence indicates that epithelial barrier function is compromised in asthma, a Th2-dominant disease, but the mechanisms involved are not well understood. The purpose of this study was to investigate the role of Th2-type cytokines on airway epithelial barrier function. METHODS: 16HBE14o- human bronchial epithelial cells monolayers were grown on collagen coated Transwell inserts. The basolateral or apical surfaces of airway epithelia were exposed to human interleukin-4 (hIL-4), TSLP, IL-25, or IL-33 alone or in combination at various concentrations and time points. We analyzed epithelial apical junctional complex (AJC) function by measuring transepithelial electrical resistance (TEER) and permeability to sodium fluorescein (NaF) over time. RESULTS: Transepithelial resistance was significantly decreased after basolateral (but not apical) exposure to IL-4 at 48 and 72 hours, with optimal IL-4 concentrations between 5-50 ng/ml. IL-4 also induced increased epithelial permeability as shown by increased apical- to basal flux of NaF. None of the other cytokines examined significantly affected TEER. Wortmannin, an inhibitor of PI3 kinase signaling pathway, did not block Il-4 mediated TEER reduction. CONCLUSIONS: Our study indicates that IL-4 has a disruptive effect on airway epithelial barrier function. IL-4 induced epithelial barrier dysfunction may contribute to airway inflammation in allergic asthma. The molecular mechanisms by which IL-4 increased bronchial epithelial cell permeability require further investigation.
817
Mutants of the Major Cockroach Allergen, Bla g 2, Modulate T Cell Responses in Cockroach-Allergic Subjects P. W. Wright1, J. Glesner2, M. Chapman2, A. Pomes2, J. Woodfolk1; 1 University of Virginia, Charlottesville, VA, 2Indoor Biotechnologies Inc., Charlottesville, VA. RATIONALE: The majority of cockroach (CR) allergic subjects have IgE antibodies to Bla g 2. Our objective was to test whether variants of Bla g 2 that contain mutations in conformational epitopes have the potential to modulate T cell responses and provide candidates for immunotherapy. METHODS: PBMCs from CR-allergic subjects (6 anti-Bla g 2 IgE antibodies) were stimulated with 12 Bla g 2 mutants (containing up to 3 point mutations), most of which showed reduced monoclonal antibody and IgE antibody binding. Cytokine responses were assessed by intracellular staining with flow cytometry, and by analysis of culture supernatants by cytometric bead assay. T cell proliferation was assessed by [3H] thymidine incorporation. RESULTS: Both wildtype Bla g 2 and mutants induced IFN-g+CD4+ T cells, with few IL-4+ or IL-10+ cells, regardless of sensitivity to Bla g 2. In cultures from Bla g 2-sensitized subjects, the deglycosylated variant N268Q enhanced secretion of IFN-g, IL-5, and IL-13. This mutant also increased TNF-a secretion, and the proportion of IFN-g+ T cells co-expressing TNF-a. These effects were generally diminished when additional mutations were introduced. In contrast to other cytokines, the variable profile of IL-10 secretion induced by different mutants (likely APC-derived) was remarkably consistent among subjects. Mutants inducing the highest proliferation contained substitutions that mapped to discrete molecular regions, but did not always induce the highest cytokine release, indicating the capacity to uncouple T-cell proliferation and cytokine pathways. CONCLUSIONS: Bla g 2 mutants modulate a Th1-dominated response and have the potential to enhance immunogenicity with or without inducing pro-inflammatory cytokines.
IgG Fc Receptor Activity in vivo is Under Complement Control E. Y. Wu, H. Jiang, G. Hester, M. M. Frank; Duke University Medical Center, Durham, NC. RATIONALE: Immunoglobulin G (IgG) receptors (FcgR) possess a critical role in the pathogenesis of immune-complex (IC) diseases. Previous reports suggest complement does not affect IC interaction with FcgR, but we assert the classical complement pathway down-regulates IC binding to FcgR. The Arthus reaction is a model of IC-mediated vasculitis, and we performed reverse passive Arthus reactions (RPAR) in normal and C1q-deficient (C1q-/-) C57BL/6 mice. We hypothesized the absence of C1q would enhance IC-mediated tissue damage. METHODS: Sedated and shaved mice (9 normal, 10 C1q-/-) were injected intradermally with 20 ml containing PBS alone on one side and affinity purified rabbit anti-BSA IgG 5 mg on the opposing side. Immediately after, purified BSA 100 mg and 125Iodine-labeled BSA 1.25 mg in PBS containing 1% Evans blue was injected intravenously. After 4 hours, skin sections were weighed and radioactivity was measured in cpm/gm of tissue. An Arthus index (AI) for each mouse was calculated by dividing cpm/gm of treated skin by cpm/gm of control skin. The Mann-Whitney U test was used to evaluate for significant difference between the median AI of the two mice groups. RESULTS: C1q-/- mice exhibited more vigorous RPAR (mean AI 3.660.8) than normal mice (mean AI 2.561.3). The median AI for C1q-/- mice was significantly higher than normal mice (3.8 vs 2.3, two-tail P<0.05). CONCLUSIONS: Our results provide physiologic evidence that the classical complement pathway down-regulates in vivo IC and FcgR mediated inflammation. They may also further understanding of why individuals with classical pathway defects are susceptible to developing autoimmune disease.
818
819
Effects of Vitamin D Deficiency on Inner City Children and Adolescents with HIV P. Poowuttikul, E. Secord; Children’s Hospital of Michigan, Wayne State University, Detroit, MI. RATIONALE: We previously reported high incidence of vitamin D deficiency in our HIV patients (primarily African American and living in low sunlight environment). We sought to identify any correlation with HIV disease markers and medications. METHODS: Serum 25 hydroxyvitamin D (25 OHD) was obtained from 160 HIV infected youth (25 OHD < 20 ng/mL classified as vitamin D deficiency; 20-35 ng/mL classified as vitamin D insufficiency). 25 OHD levels were correlated to HIV RNA (VL) and CD4+ T-cells. RESULTS: 152/160 (95%) youth had 25 OHD insufficiency. Out of these, 110/160 (68.8%) had deficiency. 24/110 (21.8%) of 25 OHD deficiency patients had undetectable VL as opposed to 15/50 (30%) with 25 OHD > 20 (p-value 5 0.15). CD4+ T-cell count was not significantly different between youth with insufficiency vs deficiency. Treatment with tenofovir or efavirenz significantly increased probability of vitamin D deficiency (48/66; 72.7% vs 27/49; 55.1%; p-value 5 0.074). We did not have sufficient youth with HIV and normal vitamin D levels to make any comparisons. CONCLUSIONS: Very high prevalence of vitamin D deficiency was confirmed. Higher 25 OHD tended to predict higher viral loads. No effect on CD4 + T cell count was observed. Treatment with tenofovir or efavirenz significantly increased the chance of vitamin D deficiency, as previously reported. HIV infection in African American youth in low sunlight areas are at significant risk for low vitamin D. Vitamin D supplementation should be routine in this population.
TUESDAY
816