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484 IL-13 but not IL-4 prolongs eosinophil survival and induces eosinophil chemotaxis
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 1, PART 3
Abstracts
481
R e d u c t i o n o f Exposure to L a b o r a t o r y Animal A l l e r g e n s s Gordon (PhD~'. J Wallace2~ J A Welch I. A Cook (MSc)I.M Jones (PhDP, R D Tee (PhDP and A J Newman Taylor (FRCP. FFGM) ~ ) National Heart and Lung Institute, London, U.K. and 2 Institute of Cancer Research, Royal Marsden Hospital, Sutton, Surrey, U.K. The prevalence of allergy to laboratory animals is related to the intensity of exposure to animal allergens (Occup Env Mad 1994;51:589). Whilst airborne animal allergen concentrations may be influenced by the litter type, cage design and stock density (Br J Ind Mad 1992;49:416), the effectiveness of methods to reduce personal exposure has not been assessed. Air samples were collected at 2 L/rain onto polytetrafluoroethylene filters and the rat urinary aeroallergen (RUA) and mouse urinary aeroallergen (MUA) concentration measured by RAST inhibition. When the animals were housed in ventilated cages (Thoren Maxlmiser system) the static MUA concentration (n - 24, median ffi 0.09 #g/m ~) was reduced 7-fold when compared with conventional cage systems (n ffi 12, median ffi 0.67 pg/m~, p<0.001). During cleaning out, personal exposure to RUA was reduced 2-fold when soiled litterwas removed by vacuum (n ffi 15, median = 25.08 #g/m ~) when compared to tipping (n = 9, median -- 42.93 #glm ~, p=0.017). The RU'A exposure associated with handling rats was reduced 25-fold when performed in a ventilated cabinet (n = 8, median = 1.19/~g/m3) compared with handling of rats on an open bench (n = 17, median := 54.39 pg/m 3, p=0.0001). The reduction of exposure to animal allergens can now effectively be achieved for tasks traditionally associated with high levels of allergen exposure.
483
482
E n d o t o x i n L e v e l s in V a r i o u s H o u s e D u s t S a m p l e s . P. Brock "f~itiiams PhD. Lenexa, KS Endutoxm (E') is a class of biotogtca[ molecules derived fi'om the outer membrane of Gram negat,ve bacteria. It can induce respirator" distress, fever, cough, shock and death Inhalation experiments demonstrate acute bronchoconstnctive resutts in asthmatics and ainvay im'iammmion in normal subjects. These effecls may be due to E's ability to induce TNF-aipha and PAF upon inhalation. E can induce mediator release from basophils and mast cells directly but also Is s3'nergmuc with allergen induced release. E also is a potent adjuvant for immunological
484
Using the chromc,~cnic modificmion of the L.imulus ly~ate assay (Biow,~iraker, I~-') in a quantitative kinetic format, we assessed 56 dust samples for E cnnlent Samples were obtained by vacuuming and large debrie removed using a 300uM mesh screen. All dilutions & extractions were with pyrogen free water at I: l0 w/v for 2 hours followed by vigorous vonexing. Controls for enhancement and inhibition were performed using 0 05 n~'ml spiking, fhe assay was linear fi-om 500 tg to 5 n~;'ml o r e with CV's below 10%. All samples were mn m duplicate. Results ranged fi'om O-62ug/gm of dust with three samples :- IOu~/ml, $ samples > 5ug/~qn and 41 ~mples>l u~;gm. Fifteen samples were below I ug/gm. Due to its proven enhancement of antigcnicity and multiple physiologic effects on asthma, the amount of endotoxin should be of serious medical concern in indoor dust samples, both with respect to the reduction and worsening of symptoms in homes inhabited by asthmatic patients.
303
GM-CSF Regulates the Functional Adhesive State of V L A - 4 E x p r e s s e d b y E o s i n o p h i l s D H Broide, MD; L Yang~ M Elices, Phl): G Jim P Sriramarao. PhD" K-L P Sunm PhD San Diego, CA As VLA-4 can exist in different functional states, we have sought to determine whether GM-CSF could regulate the functional state of eosinophil expressed VLA-4. Using a micropipette single cell adhesion assay able to measure the strength of adhesion forces, eosinophils exhibited low levels of basal adhesion to unstimulated endothelium (separation stress 0.022 .e 0.003 mdynes, mean --. SEM). In contrast, individual eosinophils bound to IL-11$ stimulated endothelium (0.49 +- 0.02 mdynes), TNF stimulated endothelium (0.62 + 0.05 mdynes) or IL4 stimulated endothelium (0.11 --. 0.01 mdynes) with increased affinity. The strength of eosinophil adhesion to CS-1 (0.18 mdynes) was greater than the strength of eosinophil adhesion to unstimulated endothelium (0.02 mdynes), but less than the strength of eosinophil adhesion to VCAM (0.31 ± 0.02 mdynes), or to IL113stimulated endothefium (0.49 ± 0.02 mdynes). After incubating eosinophils for 30 minutes with GM-CSF, the mean adhesion strength of eosinophils to CS-1 and VCAM increased significantly by 84% and 54% respectivelycompared to controls. This increased binding of eosinophils to VCAM or CS-1 was not due to alterationsin VLA-4 receptor number (assessed by FACS analysis) or alterations in VLA-4 receptor distribution (assessed by coufocal microscopy). These studies suggest that cytokines such as GM-CSF have the potential to alter the functional state of oosinophil expressed VLA-4 from a low affinity state (rolling receptor) to a high affinity state (firm adhesion receptor).
IL-13 but not IL-4 Prolongs Eosinophil Survival and Induces Eosinophil Chemotaxis. S Horie MD. Y O k u b o M D . M H o s s a i n M S . E Sato M D . H N o m u r a M D . S K o v a m a M D . M SekiLmchi M D . M a t s u m o t o , Japan. IL-4 and IL-I 3 participate in eosinophil adhesion by inducing VCAMI on endothelial cells. However, effects of these cytokines on cosinophils are unclear. Therefore, we examined the effects of IL-4 and IL-13 on survival, degranulation, and chemotactic activity of human eosinophils. Human eosinophils from normal volunteers were cultured with II.-4 or IL-13 for 4 days. IL-13 prolonged eosinophil survival in a dose-dependent manner above 3ng/ml. In addition, eosinophils cultured with IL-13 showed a reduction of DNA fragmentation. On the other hand, IL-4 showed no effects. Eosinophil survival induced by IL-13 was inhibitcd with monoclonal antibodies against IL-3 (p<0.01) and GM-CSF (p<0.05). These results suggest that IL-13 induced IL-3 and GM-CSF production from eosinophils and an autocrine mechanism is responsible for the prolonged survival. The effects of IL-13 on eosinophil chemotactic activity were also examined. When IL-13 was present in the lower compartments of Boyden chamber, IL- 13 induced chemotactic activity in a dose-dependent manner reaching a plateau at 125ng/ml. Maximal chemotactic activity of IL-13 was 65% of that of 10-TM platelet activating factor (PAF) used as a positive control. Checkerboard analysis revealed that eosinophil migration was dependent on the concentration gradient, confirming that 1L-13 is a chemotactic factor. IL-4 did not show chemotactic activity. On the degranulation of oosinophils induced by GM-CSF, PAF, or immobilized human IgG, neither IL-13 nor IL-4 showed any effects. These results suggest that IL-13 may play important roles in the survival and recruitment of eosinophils in allergic diseases.
Abstracts
481
R e d u c t i o n o f Exposure to L a b o r a t o r y Animal A l l e r g e n s s Gordon (PhD~'. J Wallace2~ J A Welch I. A Cook (MSc)I.M Jones (PhDP, R D Tee (PhDP and A J Newman Taylor (FRCP. FFGM) ~ ) National Heart and Lung Institute, London, U.K. and 2 Institute of Cancer Research, Royal Marsden Hospital, Sutton, Surrey, U.K. The prevalence of allergy to laboratory animals is related to the intensity of exposure to animal allergens (Occup Env Mad 1994;51:589). Whilst airborne animal allergen concentrations may be influenced by the litter type, cage design and stock density (Br J Ind Mad 1992;49:416), the effectiveness of methods to reduce personal exposure has not been assessed. Air samples were collected at 2 L/rain onto polytetrafluoroethylene filters and the rat urinary aeroallergen (RUA) and mouse urinary aeroallergen (MUA) concentration measured by RAST inhibition. When the animals were housed in ventilated cages (Thoren Maxlmiser system) the static MUA concentration (n - 24, median ffi 0.09 #g/m ~) was reduced 7-fold when compared with conventional cage systems (n ffi 12, median ffi 0.67 pg/m~, p<0.001). During cleaning out, personal exposure to RUA was reduced 2-fold when soiled litterwas removed by vacuum (n ffi 15, median = 25.08 #g/m ~) when compared to tipping (n = 9, median -- 42.93 #glm ~, p=0.017). The RU'A exposure associated with handling rats was reduced 25-fold when performed in a ventilated cabinet (n = 8, median = 1.19/~g/m3) compared with handling of rats on an open bench (n = 17, median := 54.39 pg/m 3, p=0.0001). The reduction of exposure to animal allergens can now effectively be achieved for tasks traditionally associated with high levels of allergen exposure.
483
482
E n d o t o x i n L e v e l s in V a r i o u s H o u s e D u s t S a m p l e s . P. Brock "f~itiiams PhD. Lenexa, KS Endutoxm (E') is a class of biotogtca[ molecules derived fi'om the outer membrane of Gram negat,ve bacteria. It can induce respirator" distress, fever, cough, shock and death Inhalation experiments demonstrate acute bronchoconstnctive resutts in asthmatics and ainvay im'iammmion in normal subjects. These effecls may be due to E's ability to induce TNF-aipha and PAF upon inhalation. E can induce mediator release from basophils and mast cells directly but also Is s3'nergmuc with allergen induced release. E also is a potent adjuvant for immunological
484
Using the chromc,~cnic modificmion of the L.imulus ly~ate assay (Biow,~iraker, I~-') in a quantitative kinetic format, we assessed 56 dust samples for E cnnlent Samples were obtained by vacuuming and large debrie removed using a 300uM mesh screen. All dilutions & extractions were with pyrogen free water at I: l0 w/v for 2 hours followed by vigorous vonexing. Controls for enhancement and inhibition were performed using 0 05 n~'ml spiking, fhe assay was linear fi-om 500 tg to 5 n~;'ml o r e with CV's below 10%. All samples were mn m duplicate. Results ranged fi'om O-62ug/gm of dust with three samples :- IOu~/ml, $ samples > 5ug/~qn and 41 ~mples>l u~;gm. Fifteen samples were below I ug/gm. Due to its proven enhancement of antigcnicity and multiple physiologic effects on asthma, the amount of endotoxin should be of serious medical concern in indoor dust samples, both with respect to the reduction and worsening of symptoms in homes inhabited by asthmatic patients.
303
GM-CSF Regulates the Functional Adhesive State of V L A - 4 E x p r e s s e d b y E o s i n o p h i l s D H Broide, MD; L Yang~ M Elices, Phl): G Jim P Sriramarao. PhD" K-L P Sunm PhD San Diego, CA As VLA-4 can exist in different functional states, we have sought to determine whether GM-CSF could regulate the functional state of eosinophil expressed VLA-4. Using a micropipette single cell adhesion assay able to measure the strength of adhesion forces, eosinophils exhibited low levels of basal adhesion to unstimulated endothelium (separation stress 0.022 .e 0.003 mdynes, mean --. SEM). In contrast, individual eosinophils bound to IL-11$ stimulated endothelium (0.49 +- 0.02 mdynes), TNF stimulated endothelium (0.62 + 0.05 mdynes) or IL4 stimulated endothelium (0.11 --. 0.01 mdynes) with increased affinity. The strength of eosinophil adhesion to CS-1 (0.18 mdynes) was greater than the strength of eosinophil adhesion to unstimulated endothelium (0.02 mdynes), but less than the strength of eosinophil adhesion to VCAM (0.31 ± 0.02 mdynes), or to IL113stimulated endothefium (0.49 ± 0.02 mdynes). After incubating eosinophils for 30 minutes with GM-CSF, the mean adhesion strength of eosinophils to CS-1 and VCAM increased significantly by 84% and 54% respectivelycompared to controls. This increased binding of eosinophils to VCAM or CS-1 was not due to alterationsin VLA-4 receptor number (assessed by FACS analysis) or alterations in VLA-4 receptor distribution (assessed by coufocal microscopy). These studies suggest that cytokines such as GM-CSF have the potential to alter the functional state of oosinophil expressed VLA-4 from a low affinity state (rolling receptor) to a high affinity state (firm adhesion receptor).
IL-13 but not IL-4 Prolongs Eosinophil Survival and Induces Eosinophil Chemotaxis. S Horie MD. Y O k u b o M D . M H o s s a i n M S . E Sato M D . H N o m u r a M D . S K o v a m a M D . M SekiLmchi M D . M a t s u m o t o , Japan. IL-4 and IL-I 3 participate in eosinophil adhesion by inducing VCAMI on endothelial cells. However, effects of these cytokines on cosinophils are unclear. Therefore, we examined the effects of IL-4 and IL-13 on survival, degranulation, and chemotactic activity of human eosinophils. Human eosinophils from normal volunteers were cultured with II.-4 or IL-13 for 4 days. IL-13 prolonged eosinophil survival in a dose-dependent manner above 3ng/ml. In addition, eosinophils cultured with IL-13 showed a reduction of DNA fragmentation. On the other hand, IL-4 showed no effects. Eosinophil survival induced by IL-13 was inhibitcd with monoclonal antibodies against IL-3 (p<0.01) and GM-CSF (p<0.05). These results suggest that IL-13 induced IL-3 and GM-CSF production from eosinophils and an autocrine mechanism is responsible for the prolonged survival. The effects of IL-13 on eosinophil chemotactic activity were also examined. When IL-13 was present in the lower compartments of Boyden chamber, IL- 13 induced chemotactic activity in a dose-dependent manner reaching a plateau at 125ng/ml. Maximal chemotactic activity of IL-13 was 65% of that of 10-TM platelet activating factor (PAF) used as a positive control. Checkerboard analysis revealed that eosinophil migration was dependent on the concentration gradient, confirming that 1L-13 is a chemotactic factor. IL-4 did not show chemotactic activity. On the degranulation of oosinophils induced by GM-CSF, PAF, or immobilized human IgG, neither IL-13 nor IL-4 showed any effects. These results suggest that IL-13 may play important roles in the survival and recruitment of eosinophils in allergic diseases.