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Neutrophil apoptosis requires bax activation via calpain-1
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Abstracts
7 '~A
"Sports, Allergy and Asthma" - Three Years Experience of a Specialized Section in an Allergy Unit Miguel-Cap~o Filipe*, Andr~ Moreira§, Jos~ Luis Delgado~, Josefina Rodrigues§, Marianela Vaz§ *Allergy, Asthma and Sports Division, I"I'
Porto, Portugal §Hospital S Jo~o, Porto, Portugal ~q)epartment of Allergology and Immunology Department, H S Jo~o, Oporto Medical School, Porto, Portugal Characterization of the population seen in a specialized "Sports, Allergy and Asthma" section. We used the "USA Olympic Questionnaire" (USOC), skin prick tests, resting spirometry, methacholine challenge (PC20M) performed in the first evaluation, and also the proposed diagnosis and instituted therapy. We evaluated 44 elite athletes (6.3_+2.6 training sessions/week), 68.2% of outdoor sports modalities (soccer: 22.7%; runners 15.9%), with mean (SD) age 21.3_+6,4 years (16F and 28M). In the responses to USOC questionnaire (n=38), the major complains related with the exercise were cough (42.1%), sibilance or trouble in breathing normally (39.5%), squeeze in the chest and incapacity to deep breath (36.8%). Twelve (31.6%) had a past history of asthma, 12 (31.6%) a familiar history of allergies, 26 (68.4%) never had allergy skin tests performed in the past, 19 (50.0%) had used beta agonists before, 5 (13.1%) inhaled corticosteroids and 3 (7.9%) imunotherapy. Twelve (31.6%) had carpets in bedroom and 17 (44.7%) had pets. Two (5.3%) reported aspirin intolerance and 3 (7.9%) penicillin allergy. In 43 of 44 (97.7%) resting spirometry was normal, 12 of 26 athletes (46.2%) had a positive methacholine test and 20 of 37 (54.1%) had positive skin prick tests. The most common diagnosis was Exercise-induced Asthma (63.6%) and/or Allergic Asthma (18.2%). Most athletes received medication (including 19 inhaled corticosteroids and 27 beta agonists) and 14 (37.8%) had no information about doping and prior written permission to use some antiasthmatic drugs. Although most athletes we studied were referred due to exercise-induced respiratory symptoms, most have been previously poorly investigated and undertreated. Bronchial hyperreactivity and atopy should be excluded in athletes with respiratory and allergic symptoms. Athletes with asthma must be informed about doping. Immunoallergology can contribute to the field of Sports Medicine, as a proper diagnosis and treatment will enable asthmatic or allergic athletes to compete effectively, without disadvantages in their sport practice.
775 Neutrophi, Apoptosis Requires Dax Activation via Calpain-, Frank Altznauer, Olivier Thomet, Hans-Uwe Simon University of Bern, Bern, Switzerland Neutrophils are important effector cells in immunity to microorganisms, particularly bacteria. In the absence of inflammation, neutrophils rapidly die via apoptosis in vivo. Similarly, neutrophils rapidly undergo apoptosis in the absence of survival factors in vitro, a process, which is called spontaneous apoptosis. Spontaneous neutrophil apoptosis is enhanced by stimulation with some members of the TNF-superfamily. Here we report, that spontaneous and Fas receptor (Fas)-induced apoptosis of human neutrophils is blocked by inhibition of the neutral cystein proteases calpain- 1 and calpain-2. Preincubation of neutrophils with calpain inhibitors prevented ethidium bromide uptake, phosphatidylserine exposure, and DNA fragmentation, suggesting an important role of ca/pains within the apoptotic pathway in neutrophils. Moreover, the induction of neutrophil apoptosis was associated with activation of ca/pain-i, but not ca/pain-2, via the degradation of their endogeneous inhibitor calpastatin. Degradation of calpastatin was caspases-dependent, as revealed by preincubating of the cells with either an inhibitory peptide of caspase-8 or a broad caspase-inhibitor. Activated ca/pain-1 cleaved the fulllength pro-apoptotic bcl-2 family-member bax (21 kDa) into a 18-kDa bax
J ALLERGYCLIN IMMUNOL JANUARY 2002
fragment, which correlated with the amount of neutrophil apoptosis. The generation of the 18-kDa bax fragment was blocked in the presence of a specific calpain- 1 inhibitor, both with or without Fas activation. The 18-kDa bax fragment is assumed to be involved in the release of cytochrome c from mitochondria, suggesting that calpain-I would be part of an apoptotic pathway proximal to mitochondria. Indeed, neutrophil apoptosis, either spontaneous or Fas-enhanced, was accompanied by the breakdown of the mitochondrial potential, which was blocked by inhibition of calpain- 1. Consistent with this model, a post-mitochondrial event, the activation ofcaspase-3, was prevented by calpain-l inhibition in both spontaneous and Fas-stimulated neutrophils. These results suggest that calpain-1 represents a key molecule within the apoptotic death pathway that act between initiator caspases and mitochondria activation, which may involve integration of a cleaved 18-kDa bax fragment into the outer mitochondrial membrane.
' ~ [ ~ Distinctive Clinical and Laboratory Features of Autosomal-
7 JF IiJFRecessive HyperlgM Syndrome
Stephen Gerard Shaw, Rebecca H Buckle~; Joseph Roberts Duke University Medical Center, Durham, NC HyperlgM syndrome (HIM) is a rare primary immunodeficiency characterized by normal or elevated serum IgM, but low or absent serum IgG, IgA and IgE levels. Recently, defects in a B cell-specific cytosolic protein, activation-induced cytidine deaminase (AID), were discovered in patients having the autosomal-recessive form of HIM. DNA for mutation analysis was available for 15 patients evaluated for HIM at our institution. Six of these patients were CD40L deficient as assessed by CD40L protein expression and genomic DNA sequencing. Of the remaining 9 patients, five (2 F; 3 M) were homozygous for the same mutation of the AID gene. This G-to-A transition (W84X) in exon 3 yields a truncated protein lacking the sequence predicted to have deaminase activity. This activity.is central to the putative RNA editing function of AID. These 5 individuals belong to 3 distinct families, but are all members of a population of North Carolina American Indians. Review of the clinical courses of this group and of the CD40L-deficient patients revealed several potentially distinguishing characteristics. As has been reported by others, lymphadenopathy, which was absent in our group of 6 XHIM patients, was frequently encountered in AID-deficient HIM patients (3/5). While XHIM patients were predisposed to opportunistic infections, and Pneumocystis carinii pneumonia in particular (4/6), AID-deficient HIM patients were not (0/5). Neutropenia was encountered in all 6 of the CD40L-deficient patients, but in none of the AID-deficient patients. Serum IgM levels were significantly higher for the AID-deficient patients (GM=941; CI=[570-1560]) than the XHIM patients (GM=79; CI=[34-182]; p=.001). Serum IgG, IgA, and IgE levels were uniformly low in both groups. B cell class switch recombination, as assessed by measuring immunoglobulin production following anti-CD40 and IL-4 stimulation in vitro, was normal in the CD40L-deficient patients, but absent in the AIDdeficient patients. Mutation analysis of the 4 other individuals (3 F; 1 M) evaluated for HIM at our institution failed to demonstrate a defective AID or CD40L gene. These individuals' B cells were capable of class switch recombination after stimulation with anti-CD40 and IL-4. In summary, we report 5 AID-deficient individuals in a series of 15 HIM patients with findings that are potentially useful when attempting to distinguish AID deficiency as a clinical entity from XHIM. This distinction is critical from a management standpoint, because early bone marrow transplantation is recommended for CD40L-deficient individuals, who often experience opportunistic infections and have a shortened life expectancy. Indeed, the clinical courses of these AID-deficient individuals, with all receiving aggressive antibiotic and IVIg therapy, suggest a better prognosis for these patients.
Abstracts
7 '~A
"Sports, Allergy and Asthma" - Three Years Experience of a Specialized Section in an Allergy Unit Miguel-Cap~o Filipe*, Andr~ Moreira§, Jos~ Luis Delgado~, Josefina Rodrigues§, Marianela Vaz§ *Allergy, Asthma and Sports Division, I"I'
Porto, Portugal §Hospital S Jo~o, Porto, Portugal ~q)epartment of Allergology and Immunology Department, H S Jo~o, Oporto Medical School, Porto, Portugal Characterization of the population seen in a specialized "Sports, Allergy and Asthma" section. We used the "USA Olympic Questionnaire" (USOC), skin prick tests, resting spirometry, methacholine challenge (PC20M) performed in the first evaluation, and also the proposed diagnosis and instituted therapy. We evaluated 44 elite athletes (6.3_+2.6 training sessions/week), 68.2% of outdoor sports modalities (soccer: 22.7%; runners 15.9%), with mean (SD) age 21.3_+6,4 years (16F and 28M). In the responses to USOC questionnaire (n=38), the major complains related with the exercise were cough (42.1%), sibilance or trouble in breathing normally (39.5%), squeeze in the chest and incapacity to deep breath (36.8%). Twelve (31.6%) had a past history of asthma, 12 (31.6%) a familiar history of allergies, 26 (68.4%) never had allergy skin tests performed in the past, 19 (50.0%) had used beta agonists before, 5 (13.1%) inhaled corticosteroids and 3 (7.9%) imunotherapy. Twelve (31.6%) had carpets in bedroom and 17 (44.7%) had pets. Two (5.3%) reported aspirin intolerance and 3 (7.9%) penicillin allergy. In 43 of 44 (97.7%) resting spirometry was normal, 12 of 26 athletes (46.2%) had a positive methacholine test and 20 of 37 (54.1%) had positive skin prick tests. The most common diagnosis was Exercise-induced Asthma (63.6%) and/or Allergic Asthma (18.2%). Most athletes received medication (including 19 inhaled corticosteroids and 27 beta agonists) and 14 (37.8%) had no information about doping and prior written permission to use some antiasthmatic drugs. Although most athletes we studied were referred due to exercise-induced respiratory symptoms, most have been previously poorly investigated and undertreated. Bronchial hyperreactivity and atopy should be excluded in athletes with respiratory and allergic symptoms. Athletes with asthma must be informed about doping. Immunoallergology can contribute to the field of Sports Medicine, as a proper diagnosis and treatment will enable asthmatic or allergic athletes to compete effectively, without disadvantages in their sport practice.
775 Neutrophi, Apoptosis Requires Dax Activation via Calpain-, Frank Altznauer, Olivier Thomet, Hans-Uwe Simon University of Bern, Bern, Switzerland Neutrophils are important effector cells in immunity to microorganisms, particularly bacteria. In the absence of inflammation, neutrophils rapidly die via apoptosis in vivo. Similarly, neutrophils rapidly undergo apoptosis in the absence of survival factors in vitro, a process, which is called spontaneous apoptosis. Spontaneous neutrophil apoptosis is enhanced by stimulation with some members of the TNF-superfamily. Here we report, that spontaneous and Fas receptor (Fas)-induced apoptosis of human neutrophils is blocked by inhibition of the neutral cystein proteases calpain- 1 and calpain-2. Preincubation of neutrophils with calpain inhibitors prevented ethidium bromide uptake, phosphatidylserine exposure, and DNA fragmentation, suggesting an important role of ca/pains within the apoptotic pathway in neutrophils. Moreover, the induction of neutrophil apoptosis was associated with activation of ca/pain-i, but not ca/pain-2, via the degradation of their endogeneous inhibitor calpastatin. Degradation of calpastatin was caspases-dependent, as revealed by preincubating of the cells with either an inhibitory peptide of caspase-8 or a broad caspase-inhibitor. Activated ca/pain-1 cleaved the fulllength pro-apoptotic bcl-2 family-member bax (21 kDa) into a 18-kDa bax
J ALLERGYCLIN IMMUNOL JANUARY 2002
fragment, which correlated with the amount of neutrophil apoptosis. The generation of the 18-kDa bax fragment was blocked in the presence of a specific calpain- 1 inhibitor, both with or without Fas activation. The 18-kDa bax fragment is assumed to be involved in the release of cytochrome c from mitochondria, suggesting that calpain-I would be part of an apoptotic pathway proximal to mitochondria. Indeed, neutrophil apoptosis, either spontaneous or Fas-enhanced, was accompanied by the breakdown of the mitochondrial potential, which was blocked by inhibition of calpain- 1. Consistent with this model, a post-mitochondrial event, the activation ofcaspase-3, was prevented by calpain-l inhibition in both spontaneous and Fas-stimulated neutrophils. These results suggest that calpain-1 represents a key molecule within the apoptotic death pathway that act between initiator caspases and mitochondria activation, which may involve integration of a cleaved 18-kDa bax fragment into the outer mitochondrial membrane.
' ~ [ ~ Distinctive Clinical and Laboratory Features of Autosomal-
7 JF IiJFRecessive HyperlgM Syndrome
Stephen Gerard Shaw, Rebecca H Buckle~; Joseph Roberts Duke University Medical Center, Durham, NC HyperlgM syndrome (HIM) is a rare primary immunodeficiency characterized by normal or elevated serum IgM, but low or absent serum IgG, IgA and IgE levels. Recently, defects in a B cell-specific cytosolic protein, activation-induced cytidine deaminase (AID), were discovered in patients having the autosomal-recessive form of HIM. DNA for mutation analysis was available for 15 patients evaluated for HIM at our institution. Six of these patients were CD40L deficient as assessed by CD40L protein expression and genomic DNA sequencing. Of the remaining 9 patients, five (2 F; 3 M) were homozygous for the same mutation of the AID gene. This G-to-A transition (W84X) in exon 3 yields a truncated protein lacking the sequence predicted to have deaminase activity. This activity.is central to the putative RNA editing function of AID. These 5 individuals belong to 3 distinct families, but are all members of a population of North Carolina American Indians. Review of the clinical courses of this group and of the CD40L-deficient patients revealed several potentially distinguishing characteristics. As has been reported by others, lymphadenopathy, which was absent in our group of 6 XHIM patients, was frequently encountered in AID-deficient HIM patients (3/5). While XHIM patients were predisposed to opportunistic infections, and Pneumocystis carinii pneumonia in particular (4/6), AID-deficient HIM patients were not (0/5). Neutropenia was encountered in all 6 of the CD40L-deficient patients, but in none of the AID-deficient patients. Serum IgM levels were significantly higher for the AID-deficient patients (GM=941; CI=[570-1560]) than the XHIM patients (GM=79; CI=[34-182]; p=.001). Serum IgG, IgA, and IgE levels were uniformly low in both groups. B cell class switch recombination, as assessed by measuring immunoglobulin production following anti-CD40 and IL-4 stimulation in vitro, was normal in the CD40L-deficient patients, but absent in the AIDdeficient patients. Mutation analysis of the 4 other individuals (3 F; 1 M) evaluated for HIM at our institution failed to demonstrate a defective AID or CD40L gene. These individuals' B cells were capable of class switch recombination after stimulation with anti-CD40 and IL-4. In summary, we report 5 AID-deficient individuals in a series of 15 HIM patients with findings that are potentially useful when attempting to distinguish AID deficiency as a clinical entity from XHIM. This distinction is critical from a management standpoint, because early bone marrow transplantation is recommended for CD40L-deficient individuals, who often experience opportunistic infections and have a shortened life expectancy. Indeed, the clinical courses of these AID-deficient individuals, with all receiving aggressive antibiotic and IVIg therapy, suggest a better prognosis for these patients.