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Th17 Related Cytokines Promote The Production of Profibrotic Cytokines from Human Eosinophils
AB366 Abstracts
L8
A Distinct Proteome Expression Profile and Extensive Changes in Cysteinyl S-Nitrosylation (SNO) in Eosinophilic Esophagitis C. M. Davis1, J. E. Wiktorowicz2, K. V. Soman2, C. Straub2, C. Nance1, M. D’Souza1, M. Lester1, S. Stafford2, K. Pazdrak2, K. Thakkar1, A. P. Olive1, A. Kurosky2; 1Baylor College of Medicine, Houston, TX, 2The University of Texas Medical Branch, Galveston, TX. RATIONALE: Inflammatory mediators have been discovered in eosinophilic esophagitis (EoE) through mRNA expression, but protein expression and post-translational modifications have not been explored. Cysteinyl S-nitrosylation (SNO) is a major mechanism through which nitric oxide modulates protein activity. Here we describe our results using a novel method (SNOFlo) to quantify SNO1 in tissues of an EoE patient compared to normal. METHODS: Proteins from biopsy samples of EoE and controls were assayed for total cysteine and half-cystine content by amino acid analysis, and fluorescent labeled before and after ascorbate treatment using the SNOFlo method. Labeled proteins were separated by 2D electrophoresis, imaged, and analyzed by SameSpots software (Nonlinear Dynamics). S-nitrosylation was determined for each protein spot, and selected spots were picked, trypsin digested, and analyzed by MALDI-TOF (AB 4800). Protein identification utilized MASCOT applied to the Swiss-Prot database. RESULTS: Applying SNOFlo to the EoE proteome, we detected differentially S-nitrosylated proteins between EoE and normal samples. Proteins from distal esophageal (DE) samples had more striking post-translational modifications than proximal (PE) tissues. Compared to controls, DE samples had 33 increased and 38 decreased SNO proteins, and PE samples had 22 increased and 27 decreased SNO proteins. Types I and II keratin, annexin, glutathione S-transferase, peroxiredoxin 1 and 2, epidermal fatty acid binding protein, and cystatin A were significantly differentially _0.001). nitrosylated in EoE (MS expectation scores < CONCLUSIONS: Evidence of unique protein expression patterns in EoE with widespread involvement of SNO implicates a possible aberrant nitric oxide regulation in disease pathogenesis. 1 Wiktorowicz, J.E., et al. Biochemistry 50:5601-5614, 2011.
L9
Th17 Related Cytokines Promote The Production of Profibrotic Cytokines from Human Eosinophils R. Halwani1, S. Al-Muhsen1, Q. Hamid2; 1King Saud University, Riyadh, SAUDI ARABIA, 2Meakins-Christie Laboratories, McGill University, Montreal, QC, CANADA. RATIONALE: Asthma is a chronic inflammatory disorder of the lung airways that is associated with airway remodeling and hyperresponsiveness. One of the most critical structural changes that affect airway functionality is fibrotic tissue deposition within the airway wall. Eosinophils have been proposed in different studies to contribute to the production of several mediators and cytokines, including the profibrotic cytokines, TGF-b and IL-11. In this study, we hypothesize that cytokines prevailing in asthmatic tissue such as Th1, Th2, and Th17 cytokines, may induce eosinophils to produce pro-fibrotic cytokines. METHODS: Eosinophils were isolated from peripheral blood of 6 mild asthmatics and 6 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and production of pro-fibrotic cytokines, TGF-b and IL-11, were determined using Intra-cellular cytokine detection and FACS analysis, immunohistochemistry, as well as real time PCR. RESULTS: The level of basal expression of TGF-b and IL-11 was significantly upregulated in asthmatic patients compared to healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of eosinophils derived pro-fibrotic cytokines. However, stimulating eosinophils with IL-17 resulted in the enhancement of the expression TGF-b and IL-11 in asthmatic individuals. CONCLUSIONS: The regulation of expression of pro-fibrotic cytokines within eosinophils is Th1/Th2 independent. However, IL-17 seems to regulate eosinophl profibrotic cytokine release in asthmatic patients and hence contributing to the accumulation of fibrotic tissue in asthmatic airways.
J ALLERGY CLIN IMMUNOL FEBRUARY 2012
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L-selectin Deficiency as a Novel Primary Immunodeficiency Causes Recurrent Infections D. M. Al-Zahrani1, M. K. AL-Sayegh2; 1Allergy and Immunolgy, King Abdulaziz Mdical City, Jeddah, SAUDI ARABIA, 2King Abdulaziz Oncology Center, Jeddah, SAUDI ARABIA. RATIONALE: Recruitment of leukocytes into sites of infection /inflammation involves leukocyte-to-leukocyte and leukocyte-to-endothelium interactions that required adhesion molecules and their ligands. We describe a case with L-selectin (CD62L) deficiency as a novel primary immunodeficiency disease. METHOD: One month old girl with history of fever for 5-days treated with ceftrixone intramuscularlly in private clinic. She was admitted with fever, seizure, and mild DIC. Ampicillin, cefotaxim and acyclovir were used for partially treated sepsis. She had fever recurrence with significant neutrophilia and several antibiotics were used for recurrent sepsis including anti-fungal. History of sibling death at 6-months of age due to infection and recurrent sepsis raised possibility of underlying immunodeficiency. RESULTS: Complete blood count showed: leucocytes 61,690 cells/cmm; absolute neutrophils 38,865 cells/cmm and lymphocytes 11,721 cells/ cmm, haemoglobin 15.3 g/dl and thrombocytes 27,000/cmm. Erythrocyte sedimentation rate 76mm/hr. Her coagulopathy resolved. Bone marrow biopsy: showed no malignancy. Septic screen, viral and metabolic screen were negative. Immunoglobulin levels, T and B-Lymphocyte markers were normal. Patient’s neutrophils showed absence of L-selectin (CD62L) expression and migration failure through cell culture inserts towards concentration gradient of the chemoattractant (fMLP) compared to normal control by flowcytomerty. CD11/18 and CD 15 were normal. CONCLUSION: The adhesion molecules and their ligands are all required for immune defense against microbes. Isolated L-selectin (CD62L) deficiency causing primary immunodeficiency was not previously described. We described isolated L-selectin deficiency confirmed by absence of CD62L molecule on leukocytes as a novel primary immunodeficiency that caused recurrent sepsis. L-selectin deficiency should be considered in the differential diagnosis of recurrent infections.
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Transplacental Passage of IgG anti-IgE/IgE Immune Complexes (ICs) A. P. Matson1,2, E. Rafti1; 1Univ of CT Health Ctr, Farmington, CT, 2CT Children’s Med Ctr, Hartford, CT. RATIONALE: Maternal IgG enhances the placental transfer of several antigens; however, it is unclear if maternal IgG may facilitate the transplacental passage of IgE as IgG anti-IgE/IgE ICs. METHODS: Mother/infant dyads were prenatally recruited and maternal allergic status was determined by questionnaire. Maternal blood was collected prior to delivery and cord blood (CB) was collected immediately after birth. Serum concentrations of IgG anti-IgE/IgE ICs were determined using a modified ELISA. The relationship between maternal and infant ICs was determined by Spearman’s rank correlation. Maternal and CB total IgE levels were determined by Phadia (Thermo Fisher Scientific) ImmunoCAP. IgA was measured to detect maternal blood contamination of CB. RESULTS: Serum concentrations of IgG anti-IgE/IgE ICs were determined in 30 allergic mother/infant dyads and 73 non-allergic mother/infant dyads. There was no difference in concentrations of IgG anti-IgE/IgE ICs in mothers or infants based on maternal history of allergic disease. Overall, maternal and CB concentrations of IgG anti-IgE/IgE ICs were highly correlated (rho50.89, p<0.001), suggesting placental transmission. A similar relationship was observed for the allergic (rho50.90, p<0.001) and non-allergic (rho50.88, p<0.001) groups. IgE concentrations were higher in allergic mothers compared to non-allergic mothers (p<0.01). Maternal and infant concentrations of IgG anti-IgE/IgE ICs correlated poorly with CB IgE concentrations. CONCLUSIONS: Fetal acquisition of maternal IgE in the form of IgG anti-IgE/IgE ICs likely occurs via the transplacental route. The ability of maternal IgG anti-IgE/IgE ICs to influence fetal and/or neonatal immunity remains unclear.
L8
A Distinct Proteome Expression Profile and Extensive Changes in Cysteinyl S-Nitrosylation (SNO) in Eosinophilic Esophagitis C. M. Davis1, J. E. Wiktorowicz2, K. V. Soman2, C. Straub2, C. Nance1, M. D’Souza1, M. Lester1, S. Stafford2, K. Pazdrak2, K. Thakkar1, A. P. Olive1, A. Kurosky2; 1Baylor College of Medicine, Houston, TX, 2The University of Texas Medical Branch, Galveston, TX. RATIONALE: Inflammatory mediators have been discovered in eosinophilic esophagitis (EoE) through mRNA expression, but protein expression and post-translational modifications have not been explored. Cysteinyl S-nitrosylation (SNO) is a major mechanism through which nitric oxide modulates protein activity. Here we describe our results using a novel method (SNOFlo) to quantify SNO1 in tissues of an EoE patient compared to normal. METHODS: Proteins from biopsy samples of EoE and controls were assayed for total cysteine and half-cystine content by amino acid analysis, and fluorescent labeled before and after ascorbate treatment using the SNOFlo method. Labeled proteins were separated by 2D electrophoresis, imaged, and analyzed by SameSpots software (Nonlinear Dynamics). S-nitrosylation was determined for each protein spot, and selected spots were picked, trypsin digested, and analyzed by MALDI-TOF (AB 4800). Protein identification utilized MASCOT applied to the Swiss-Prot database. RESULTS: Applying SNOFlo to the EoE proteome, we detected differentially S-nitrosylated proteins between EoE and normal samples. Proteins from distal esophageal (DE) samples had more striking post-translational modifications than proximal (PE) tissues. Compared to controls, DE samples had 33 increased and 38 decreased SNO proteins, and PE samples had 22 increased and 27 decreased SNO proteins. Types I and II keratin, annexin, glutathione S-transferase, peroxiredoxin 1 and 2, epidermal fatty acid binding protein, and cystatin A were significantly differentially _0.001). nitrosylated in EoE (MS expectation scores < CONCLUSIONS: Evidence of unique protein expression patterns in EoE with widespread involvement of SNO implicates a possible aberrant nitric oxide regulation in disease pathogenesis. 1 Wiktorowicz, J.E., et al. Biochemistry 50:5601-5614, 2011.
L9
Th17 Related Cytokines Promote The Production of Profibrotic Cytokines from Human Eosinophils R. Halwani1, S. Al-Muhsen1, Q. Hamid2; 1King Saud University, Riyadh, SAUDI ARABIA, 2Meakins-Christie Laboratories, McGill University, Montreal, QC, CANADA. RATIONALE: Asthma is a chronic inflammatory disorder of the lung airways that is associated with airway remodeling and hyperresponsiveness. One of the most critical structural changes that affect airway functionality is fibrotic tissue deposition within the airway wall. Eosinophils have been proposed in different studies to contribute to the production of several mediators and cytokines, including the profibrotic cytokines, TGF-b and IL-11. In this study, we hypothesize that cytokines prevailing in asthmatic tissue such as Th1, Th2, and Th17 cytokines, may induce eosinophils to produce pro-fibrotic cytokines. METHODS: Eosinophils were isolated from peripheral blood of 6 mild asthmatics and 6 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and production of pro-fibrotic cytokines, TGF-b and IL-11, were determined using Intra-cellular cytokine detection and FACS analysis, immunohistochemistry, as well as real time PCR. RESULTS: The level of basal expression of TGF-b and IL-11 was significantly upregulated in asthmatic patients compared to healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of eosinophils derived pro-fibrotic cytokines. However, stimulating eosinophils with IL-17 resulted in the enhancement of the expression TGF-b and IL-11 in asthmatic individuals. CONCLUSIONS: The regulation of expression of pro-fibrotic cytokines within eosinophils is Th1/Th2 independent. However, IL-17 seems to regulate eosinophl profibrotic cytokine release in asthmatic patients and hence contributing to the accumulation of fibrotic tissue in asthmatic airways.
J ALLERGY CLIN IMMUNOL FEBRUARY 2012
L10
L-selectin Deficiency as a Novel Primary Immunodeficiency Causes Recurrent Infections D. M. Al-Zahrani1, M. K. AL-Sayegh2; 1Allergy and Immunolgy, King Abdulaziz Mdical City, Jeddah, SAUDI ARABIA, 2King Abdulaziz Oncology Center, Jeddah, SAUDI ARABIA. RATIONALE: Recruitment of leukocytes into sites of infection /inflammation involves leukocyte-to-leukocyte and leukocyte-to-endothelium interactions that required adhesion molecules and their ligands. We describe a case with L-selectin (CD62L) deficiency as a novel primary immunodeficiency disease. METHOD: One month old girl with history of fever for 5-days treated with ceftrixone intramuscularlly in private clinic. She was admitted with fever, seizure, and mild DIC. Ampicillin, cefotaxim and acyclovir were used for partially treated sepsis. She had fever recurrence with significant neutrophilia and several antibiotics were used for recurrent sepsis including anti-fungal. History of sibling death at 6-months of age due to infection and recurrent sepsis raised possibility of underlying immunodeficiency. RESULTS: Complete blood count showed: leucocytes 61,690 cells/cmm; absolute neutrophils 38,865 cells/cmm and lymphocytes 11,721 cells/ cmm, haemoglobin 15.3 g/dl and thrombocytes 27,000/cmm. Erythrocyte sedimentation rate 76mm/hr. Her coagulopathy resolved. Bone marrow biopsy: showed no malignancy. Septic screen, viral and metabolic screen were negative. Immunoglobulin levels, T and B-Lymphocyte markers were normal. Patient’s neutrophils showed absence of L-selectin (CD62L) expression and migration failure through cell culture inserts towards concentration gradient of the chemoattractant (fMLP) compared to normal control by flowcytomerty. CD11/18 and CD 15 were normal. CONCLUSION: The adhesion molecules and their ligands are all required for immune defense against microbes. Isolated L-selectin (CD62L) deficiency causing primary immunodeficiency was not previously described. We described isolated L-selectin deficiency confirmed by absence of CD62L molecule on leukocytes as a novel primary immunodeficiency that caused recurrent sepsis. L-selectin deficiency should be considered in the differential diagnosis of recurrent infections.
L11
Transplacental Passage of IgG anti-IgE/IgE Immune Complexes (ICs) A. P. Matson1,2, E. Rafti1; 1Univ of CT Health Ctr, Farmington, CT, 2CT Children’s Med Ctr, Hartford, CT. RATIONALE: Maternal IgG enhances the placental transfer of several antigens; however, it is unclear if maternal IgG may facilitate the transplacental passage of IgE as IgG anti-IgE/IgE ICs. METHODS: Mother/infant dyads were prenatally recruited and maternal allergic status was determined by questionnaire. Maternal blood was collected prior to delivery and cord blood (CB) was collected immediately after birth. Serum concentrations of IgG anti-IgE/IgE ICs were determined using a modified ELISA. The relationship between maternal and infant ICs was determined by Spearman’s rank correlation. Maternal and CB total IgE levels were determined by Phadia (Thermo Fisher Scientific) ImmunoCAP. IgA was measured to detect maternal blood contamination of CB. RESULTS: Serum concentrations of IgG anti-IgE/IgE ICs were determined in 30 allergic mother/infant dyads and 73 non-allergic mother/infant dyads. There was no difference in concentrations of IgG anti-IgE/IgE ICs in mothers or infants based on maternal history of allergic disease. Overall, maternal and CB concentrations of IgG anti-IgE/IgE ICs were highly correlated (rho50.89, p<0.001), suggesting placental transmission. A similar relationship was observed for the allergic (rho50.90, p<0.001) and non-allergic (rho50.88, p<0.001) groups. IgE concentrations were higher in allergic mothers compared to non-allergic mothers (p<0.01). Maternal and infant concentrations of IgG anti-IgE/IgE ICs correlated poorly with CB IgE concentrations. CONCLUSIONS: Fetal acquisition of maternal IgE in the form of IgG anti-IgE/IgE ICs likely occurs via the transplacental route. The ability of maternal IgG anti-IgE/IgE ICs to influence fetal and/or neonatal immunity remains unclear.