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B cell apoptosis induced by gp120 in HIV-1 infected individuals is inhibited by SDF-1α
S60 Abstracts
SATURDAY
Constitutive Expression of Interleukin 14 (IL-14) in Transgenic Mice Leads to Enhanced Responses to Vaccinations and Autoimmunity C. XU1, C. Zhang1, L. Shen1, D. E. Skyes1, H. Zeng1, S. Tang1, P. D. Soloway2, S. Brooks3, S. Sharma3, R. J. Ford4, Y. Lin-Lee4, J. Horn5, L. Martin6, L. Ludwig1, P. Liang2, J. L. Ambrus, Jr.1; 1Allergy, immunology and rheumatology, SUNY at Buffalo School of Medicine and Biomedical sciences, Buffalo, NY, 2Rosewell Park Cancer Institute, Buffalo, NY, 3Surgery, SUNY at Buffalo School of Medicine and Biomedical sciences, Buffalo, NY, 4Hemato-Pathology, MD Anderson Cancer Center, Huston, TX, 5Pathology, SUNY at Buffalo School of Medicine and Biomedical sciences, Buffalo, NY, 6Allergy, immunology and rheumatology, Veterinary Services, SUNY, Buffalo, NY. RATIONALE: We have previously described human interleukin 14 (IL14) as a B cell growth factor. In vitro studies suggested that IL-14 might be important in inducing memory B cell responses. Increased expression of IL-14 was demonstrated in patients with SLE, who have memory responses to autoantigens. Transgenic mice were developed expressing IL-14 to evaluate vaccination responses and the development of autoimmunity in vivo. METHODS: IL-14 transgenic mice were developed using pESR. Transgenic mice and littermate controls were vaccinated with NP-OVA and NP-Ficoll and the specific response to NP determined. Autopsies were done to evaluate renal disease and salivary gland destruction. RESULTS: Following NP-OVA vaccination, IL-14 transgenic mice showed enhanced IgG anti-NP responses compared to littermate controls (p =0.002) The IgM anti-NP responses were not significantly different between these two groups. When NP-Ficoll was used as the immunogen only the IgM anti-NP response was significantly enhanced in the IL-14 transgenic mice compared to the littermate controls (p < 0.001). At 30 weeks of age, IL-14 transgenic mice demonstrated both elevated serum IgM and IgG compared to the littermate controls (p < 0.02 in each case). The IL-14 transgenic mice developed mild proteinuria secondary to deposition of IgG and IgM in the kidneys and lymphocytic infiltration of the parotid glands similar to that seen in Sjogren syndrome. CONCLUSIONS: IL-14 is a highly conserved cytokine that has an important role in B cell biology. When constitutively expressed in transgenic mice IL-14 causes both enhanced responses to vaccinations and autoimmunity. Funding: Oishei, Wendt, and Kaleida Health
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B Cell Apoptosis Induced by gp120 in HIV-1 Infected Individuals Is Inhibited by SDF-1 C. L. Nance1, W. T. Shearer2; 1Allergy/Immunology, Texas Childrens Hospital, Houston, TX, 2Pediatrics, Baylor College of Medicine, Houston, TX. RATIONALE: HIV-1 infection leads to the progressive loss of immune functions and cell numbers. Loss of humoral immunity correlates with B cell apoptosis. The chemokine, SDF-1, natural ligand of the HIV-1 coreceptor, CXCR4, exerts a protective effect in HIV-1 infection. We evaluated the role SDF-1 exerts on gp120-induced B cell apoptosis, through anti-apoptotic factors, Bcl-2 family members. METHODS: Purified human B cells were isolated from the peripheral blood of HIV-1-infected and uninfected subjects and activated with antiCD40 and IL4. Apoptotic events were assessed by Annexin V/PI binding and DNA fragmentation assay (TUNEL). Analysis of the apoptotic processes, Fas (CD95) and Bcl-2, were made by flow cytometric, ELISA, colorimetric, protein assays, and RT-PCR. RESULTS: Baseline Annexin V+/PI- binding was increased three-fold in B cells from HIV-1 seropositive patients compared to uninfected. In B cells from HIV-1-infected subjects, gp120 (100ng/ml) increased apoptosis 58% (p<0.01) and SDF-1 (100ng/ml) returned levels to baseline. Confirmatory data was obtained with DNA fragmentation analysis. Apo-1/Fas levels (69%) and CD95 expression (43%) were both increased (p<0.01). SDF-1 (100ng/ml) returned Fas and CD95 levels to baseline. Bcl-2
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J ALLERGY CLIN IMMUNOL FEBRUARY 2005
mRNA expression was significantly up regulated by SDF-1 (78%) (p<0.01). Bcl-xL and Bcl-2 expression was down regulated by gp120. This gp120-induced down regulation was blocked by SDF-1. Abrogation of SDF-1-induced effects occurred in the presence of an anti-SDF-1 antibody, to prove specificity. CONCLUSION: These findings suggest a possible therapeutic use for SDF-1 to reduce gp120-induced B cell apoptosis through the CXCR4dependent signaling pathway and to restore Bcl-2 function in HIV-1infected patients. Funding: NIH, NIAID AI36211, AI27551 Splenectomized Patients Make IgG Antibodies to 23-Valent Non-Conjugated Pneumococcal Vaccine Despite Having Reduced Circulating CD27+ Memory B Cells H. A. Wasserstrom1, J. B. Bussel2, L. C. L. Lim3, N. T. Guerrero3, M. H. Wissert2, L. Radigan1, C. Cunningham-Rundles1; 1Mount Sinai School of Medicine, New York, NY, 2Weill Medical College of Cornell University, New York, NY, 3Specialty Laboratories, Santa Monica, CA. RATIONALE: Splenectomized patients are believed to make poor antibody responses to polyvalent non-conjugated pneumococcal vaccines (PV). Recent reports describe reduced (CD27+) memory B cells in these patients. To verify these findings and determine if they correlate with inadequate antibody production, we measured splenectomized patients’ antibody responses to PV and enumerated their circulating B cell populations. METHODS: Thirteen patients and 10 non-splenectomized controls received PV. IgG antibody titers were measured on day0 and 4-6 weeks later. Protection was defined as post-vaccination IgG titer ≥1.3ug/ml or post:pre-vaccination titer ratio ≥4, in at least 16 of the 23 serotypes. Peripheral blood mononuclear cells of 21 patients and 10 controls were isolated, and IgM+IgD+CD27+ and IgM-IgD-CD27+ B cells were enumerated by flow cytometry. Patients had not received IVIG, Rituxan or immunosuppressive therapy within 6 months, or PV within 2 years of enrollment. No controls had ever received PV. RESULTS: Of those vaccinated, 9 patients and 9 controls achieved protective titers ( 2=1.433; p=0.339). Patient and control cohorts achieved protective titers to a mean number of 17 and 20 serotypes, respectively (p=0.134). The geometric means of each cohort’s post-vaccination titers did not differ significantly in 22 of 23 serotypes. Patients had a greater percentage of circulating B cells (patients:12.05%±7.61; controls:7.99%±2.92; =0.042), but a significantly reduced CD27+ B cell component (patients:12.38%±8.48; controls:40.46%±18.68; =0.001). This reduction was not specific to either CD27+ B cell population (IgM+IgD+CD27+: patients:31.64%±19.72; controls:44.58%±16.01; =0.081. IgM-IgDCD27+: patients:52.05±23.71; controls:48.08%±13.89; =0.628.) CONCLUSIONS: Splenectomized patients can make adequate IgG antibody responses to PV despite having reduced circulating CD27+ memory B cells. Funding: NIH
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Dog Exposure in Early Childhood Reduces Atopic Dermatitis and Wheezing at Age 3 J. D. Bufford1, C. L. Reardon1, J. Kohls1, Z. Li2, R. Gangnon2, K. A. Roberg1, C. Tisler1, E. Anderson1, D. DaSilva1, P. A. Eggleston3, R. F. Lemanske1, J. E. Gern1; 1Pediatrics, University of Wisconsin-Madison, Madison, WI, 2Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI, 3Johns Hopkins University, Baltimore, MD. RATIONALE: Exposure to dogs in the first year of life is associated with increased production of IL-10 and a decreased risk of atopic dermatitis (AD) and allergic sensitization at age 1. The effect of pet exposure on subsequent immune development and atopic diseases has not been well studied. METHODS: Pet ownership at birth was recorded for 285 children enrolled in the COAST study (Childhood Origins of ASThma). PHAinduced cytokine responses from peripheral blood mononuclear cells
242
SATURDAY
Constitutive Expression of Interleukin 14 (IL-14) in Transgenic Mice Leads to Enhanced Responses to Vaccinations and Autoimmunity C. XU1, C. Zhang1, L. Shen1, D. E. Skyes1, H. Zeng1, S. Tang1, P. D. Soloway2, S. Brooks3, S. Sharma3, R. J. Ford4, Y. Lin-Lee4, J. Horn5, L. Martin6, L. Ludwig1, P. Liang2, J. L. Ambrus, Jr.1; 1Allergy, immunology and rheumatology, SUNY at Buffalo School of Medicine and Biomedical sciences, Buffalo, NY, 2Rosewell Park Cancer Institute, Buffalo, NY, 3Surgery, SUNY at Buffalo School of Medicine and Biomedical sciences, Buffalo, NY, 4Hemato-Pathology, MD Anderson Cancer Center, Huston, TX, 5Pathology, SUNY at Buffalo School of Medicine and Biomedical sciences, Buffalo, NY, 6Allergy, immunology and rheumatology, Veterinary Services, SUNY, Buffalo, NY. RATIONALE: We have previously described human interleukin 14 (IL14) as a B cell growth factor. In vitro studies suggested that IL-14 might be important in inducing memory B cell responses. Increased expression of IL-14 was demonstrated in patients with SLE, who have memory responses to autoantigens. Transgenic mice were developed expressing IL-14 to evaluate vaccination responses and the development of autoimmunity in vivo. METHODS: IL-14 transgenic mice were developed using pESR. Transgenic mice and littermate controls were vaccinated with NP-OVA and NP-Ficoll and the specific response to NP determined. Autopsies were done to evaluate renal disease and salivary gland destruction. RESULTS: Following NP-OVA vaccination, IL-14 transgenic mice showed enhanced IgG anti-NP responses compared to littermate controls (p =0.002) The IgM anti-NP responses were not significantly different between these two groups. When NP-Ficoll was used as the immunogen only the IgM anti-NP response was significantly enhanced in the IL-14 transgenic mice compared to the littermate controls (p < 0.001). At 30 weeks of age, IL-14 transgenic mice demonstrated both elevated serum IgM and IgG compared to the littermate controls (p < 0.02 in each case). The IL-14 transgenic mice developed mild proteinuria secondary to deposition of IgG and IgM in the kidneys and lymphocytic infiltration of the parotid glands similar to that seen in Sjogren syndrome. CONCLUSIONS: IL-14 is a highly conserved cytokine that has an important role in B cell biology. When constitutively expressed in transgenic mice IL-14 causes both enhanced responses to vaccinations and autoimmunity. Funding: Oishei, Wendt, and Kaleida Health
239
B Cell Apoptosis Induced by gp120 in HIV-1 Infected Individuals Is Inhibited by SDF-1 C. L. Nance1, W. T. Shearer2; 1Allergy/Immunology, Texas Childrens Hospital, Houston, TX, 2Pediatrics, Baylor College of Medicine, Houston, TX. RATIONALE: HIV-1 infection leads to the progressive loss of immune functions and cell numbers. Loss of humoral immunity correlates with B cell apoptosis. The chemokine, SDF-1, natural ligand of the HIV-1 coreceptor, CXCR4, exerts a protective effect in HIV-1 infection. We evaluated the role SDF-1 exerts on gp120-induced B cell apoptosis, through anti-apoptotic factors, Bcl-2 family members. METHODS: Purified human B cells were isolated from the peripheral blood of HIV-1-infected and uninfected subjects and activated with antiCD40 and IL4. Apoptotic events were assessed by Annexin V/PI binding and DNA fragmentation assay (TUNEL). Analysis of the apoptotic processes, Fas (CD95) and Bcl-2, were made by flow cytometric, ELISA, colorimetric, protein assays, and RT-PCR. RESULTS: Baseline Annexin V+/PI- binding was increased three-fold in B cells from HIV-1 seropositive patients compared to uninfected. In B cells from HIV-1-infected subjects, gp120 (100ng/ml) increased apoptosis 58% (p<0.01) and SDF-1 (100ng/ml) returned levels to baseline. Confirmatory data was obtained with DNA fragmentation analysis. Apo-1/Fas levels (69%) and CD95 expression (43%) were both increased (p<0.01). SDF-1 (100ng/ml) returned Fas and CD95 levels to baseline. Bcl-2
240
J ALLERGY CLIN IMMUNOL FEBRUARY 2005
mRNA expression was significantly up regulated by SDF-1 (78%) (p<0.01). Bcl-xL and Bcl-2 expression was down regulated by gp120. This gp120-induced down regulation was blocked by SDF-1. Abrogation of SDF-1-induced effects occurred in the presence of an anti-SDF-1 antibody, to prove specificity. CONCLUSION: These findings suggest a possible therapeutic use for SDF-1 to reduce gp120-induced B cell apoptosis through the CXCR4dependent signaling pathway and to restore Bcl-2 function in HIV-1infected patients. Funding: NIH, NIAID AI36211, AI27551 Splenectomized Patients Make IgG Antibodies to 23-Valent Non-Conjugated Pneumococcal Vaccine Despite Having Reduced Circulating CD27+ Memory B Cells H. A. Wasserstrom1, J. B. Bussel2, L. C. L. Lim3, N. T. Guerrero3, M. H. Wissert2, L. Radigan1, C. Cunningham-Rundles1; 1Mount Sinai School of Medicine, New York, NY, 2Weill Medical College of Cornell University, New York, NY, 3Specialty Laboratories, Santa Monica, CA. RATIONALE: Splenectomized patients are believed to make poor antibody responses to polyvalent non-conjugated pneumococcal vaccines (PV). Recent reports describe reduced (CD27+) memory B cells in these patients. To verify these findings and determine if they correlate with inadequate antibody production, we measured splenectomized patients’ antibody responses to PV and enumerated their circulating B cell populations. METHODS: Thirteen patients and 10 non-splenectomized controls received PV. IgG antibody titers were measured on day0 and 4-6 weeks later. Protection was defined as post-vaccination IgG titer ≥1.3ug/ml or post:pre-vaccination titer ratio ≥4, in at least 16 of the 23 serotypes. Peripheral blood mononuclear cells of 21 patients and 10 controls were isolated, and IgM+IgD+CD27+ and IgM-IgD-CD27+ B cells were enumerated by flow cytometry. Patients had not received IVIG, Rituxan or immunosuppressive therapy within 6 months, or PV within 2 years of enrollment. No controls had ever received PV. RESULTS: Of those vaccinated, 9 patients and 9 controls achieved protective titers ( 2=1.433; p=0.339). Patient and control cohorts achieved protective titers to a mean number of 17 and 20 serotypes, respectively (p=0.134). The geometric means of each cohort’s post-vaccination titers did not differ significantly in 22 of 23 serotypes. Patients had a greater percentage of circulating B cells (patients:12.05%±7.61; controls:7.99%±2.92; =0.042), but a significantly reduced CD27+ B cell component (patients:12.38%±8.48; controls:40.46%±18.68; =0.001). This reduction was not specific to either CD27+ B cell population (IgM+IgD+CD27+: patients:31.64%±19.72; controls:44.58%±16.01; =0.081. IgM-IgDCD27+: patients:52.05±23.71; controls:48.08%±13.89; =0.628.) CONCLUSIONS: Splenectomized patients can make adequate IgG antibody responses to PV despite having reduced circulating CD27+ memory B cells. Funding: NIH
241
Dog Exposure in Early Childhood Reduces Atopic Dermatitis and Wheezing at Age 3 J. D. Bufford1, C. L. Reardon1, J. Kohls1, Z. Li2, R. Gangnon2, K. A. Roberg1, C. Tisler1, E. Anderson1, D. DaSilva1, P. A. Eggleston3, R. F. Lemanske1, J. E. Gern1; 1Pediatrics, University of Wisconsin-Madison, Madison, WI, 2Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI, 3Johns Hopkins University, Baltimore, MD. RATIONALE: Exposure to dogs in the first year of life is associated with increased production of IL-10 and a decreased risk of atopic dermatitis (AD) and allergic sensitization at age 1. The effect of pet exposure on subsequent immune development and atopic diseases has not been well studied. METHODS: Pet ownership at birth was recorded for 285 children enrolled in the COAST study (Childhood Origins of ASThma). PHAinduced cytokine responses from peripheral blood mononuclear cells
242