il-10 deficiency prevents il-5 overproduction and eosinophilic inflammation in a murine model of...

0014-2980/00/0202-382$17.50+.50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000

IL-10 deficiency prevents IL-5 overproduction andeosinophilic inflammation in a murine model ofasthma-like reaction

Xi Yang, Shuhe Wang, Yijun Fan and Xiaobing Han

Immune Regulation of Allergy Research Group, Departments of Medical Microbiology andImmunology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada

Eosinophilic inflammation and bronchial mucus secretion are among the characteristic path-ological changes in asthmatic reaction, which is mediated by Th2 type responses. Althoughit belongs to Th2 cytokines especially in the mouse, IL-10 is often considered an inhibitorycytokine for both Th1 and Th2 cells. In the present study, using a murine asthma modelinduced by ovalbumin (OVA), we demonstrated that endogenous IL-10 is critical for thedevelopment of asthma-like responses. Specifically, in comparison with wild-type controls,IL-10 gene knockout (KO) mice showed significantly reduced IL-5 production, eosinophilicinflammation and mucus production without notable changes in IL-4 and IgE responses fol-lowing i.p. sensitization and subsequent intranasal challenge with OVA. In addition, Th1-related cytokine (IFN- + and IL-12) production in IL-10 KO mice was significantly higher thanthat in wild-type mice. The results suggest that endogenous IL-10 plays an important role inpromoting pulmonary eosinophilic inflammatory reaction and mucus production during asth-matic reaction. The data also argue that IL-10 may be more influential in the development ofIL-5-producing Th2 cells which differ from typical Th2 cells producing both IL-4 and IL-5.

Key words: Allergy / Asthma / Cytokine / Knockout / Eosinophilia

Received 1/6/99Revised 12/8/99Accepted 18/10/99

[I 19752]

Abbreviations: KO: Gene knockout alum: Al(OH)3 BAL:Broncho-alveolar lavage H & E: Hematoxylin and eosinPCA: Passive cutaneous anaphylaxis

1 Introduction

The incidence of allergic diseases has dramaticallyincreased during past decades, especially in the devel-oped world [1, 2]. Asthma, a prevalent allergic disease, ischaracterized by airway hyperreactivity, eosinophilicinflammation, mucus secretion and, in many cases, IgEproduction. The fact that a large amount of eosinophilsare recruited into the airways during asthmatic reactionsuggests that eosinophils play an important role in air-way hyperreactivity. Indeed, eosinophils have beenshown to be able to release a variety of mediators thatalter the responsiveness of airways and damage airwaycells [3, 4]. Recent studies have clearly demonstrated theimportance of Th2 type CD4 T cells in the initiation andmediation of allergic responses, including asthma [5–9].IL-5 production appears particularly important for eosin-ophilic inflammation observed in both human and animalmodel sytems [10–14]. Thus, IL-5 gene knockout (KO)

mice showed lack of eosinophilic reaction to allergensand administration of neutralizing antibodies to IL-5inhibited helminth-induced eosinophilia [11, 12].

The role of IL-10 in allergic responses remains unclear.IL-10, although it belongs to the group of cytokines pro-duced by Th2 CD4 T cells in mouse models, has beenconsidered an inhibitory factor for allergic responses andTh2 cytokine production [15–18]. Thus, exogenousrecombinant IL-10 suppresses IL-5 production by CD4 Tcells and inhibits airway [17] and peritoneal [18] eosino-philic inflammation induced by allergen. Moreover, aller-gen immunotherapy induced IL-10 production whichpartially inhibited the release of histamine and sulfidoleu-kotriene by mast cells and basophils [19]. Paradoxically,IL-10 was also found in numerous studies to be associ-ated with IL-5 production [20–23]. The associationbetween IL-10 and IL-5 appears to be more close thanthat between IL-10 and IL-4, because IL-4-deficient micestill show significant IL-10 and IL-5 production followingSchistosoma mansoni soluble egg antigen (SEA) immu-nization [21].

To more directly examine the role played by IL-10 inatopic allergy, we studied the asthma-like reaction in

382 X. Yang et al. Eur. J. Immunol. 2000. 30: 382–391

Fig. 1. Differential cellular recruitment into the airways ofIL-10 KO and wild-type mice shown by BAL. Mice (fourmice/group) were sensitized as described in Sect. 4.2 andkilled at day 7 post intranasal challenge and the pulmonarycellular infiltration was examined by differential cell counts ofthe BAL fluids. The figure shows the absolute number ofeach infiltrating cellular population (A) and the proportion ofeach cellular component composing of the total BAL cells(B). Data are shown as mean ± SD of each group. One repre-sentative experiment of three independent experiments isshown. * p X 0.05; ** p X 0.01; *** p X 0.001, **** p X 0.0001,infiltrating cells in IL-10 KO mice compared with that in wild-type mice. ß , Total; , monocytes; lymphocytes; neu-trophils; 1 eosinophils.

IL-10 KO mice induced by OVA sensitization/challenge.The results demonstrate that, whereas wild-type miceshowed dominant airway eosinophilic infiltration, micewithout endogenous IL-10 exhibited dominant mononu-clear cell recruitment into airways following allergenexposure. Moreover, development of the mucus-secreting goblet cells within the respiratory epitheliumwas markedly reduced in IL-10 KO mice compared withwild-type controls. The changes in pulmonary inflamma-tory cells and reduction of mucus production in IL-10 KOmice were correlated with decreased IL-5 productionand increased IFN- + production without significant alter-ation in IL-4 and IgE production. Taken together, thepresent study suggests that endogenous IL-10 plays animportant role in promoting bronchial eosinophilicinflammatory reaction and goblet cell developmentinduced by allergen, mainly via enhancing IL-5 produc-tion. The data suggest that IL-10 may be particularlyimportant in the development of IL-5-producing Th2cells which may differ from typical Th2 cells producingIL-4 and IL-5.

2 Results

2.1 IL-10 KO mice show altered pulmonaryinflammatory cellular infiltration and mucusproduction

To directly examine the role of IL-10 in allergen-inducedpulmonary inflammation, we analyzed cellular compo-nents in the bronchoalveolar lavages (BAL) of OVA-sensitized IL-10 KO and wild-type mice followingantigen-specific intranasal challenge. Our preliminaryexperiments examining the kinetics of airway cellularinfiltration and serum IgE production in OVA-sensitizedwild-type and IL-10 KO mice at 2, 4, 5, 7 and 10 days fol-lowing intranasal challenge with OVA showed that airwayinfiltration was detectable at day 2 following challenge,peaking at day 5–7 and declining at day 10 while OVA-specific IgE was not measurable before day 7 followingchallenge (data not shown). To test airway reactivity andIgE production in same experiments, we chose day 7 fol-lowing intranasal challenge for analysis of airway inflam-mation, cytokine and antibody responses. As shown inFig. 1, although the total amount of cellular infiltrationwas comparable in the two groups of mice, the composi-tion of their inflammatory cells was remarkably different.Wild-type mice showed dominant eosinophilic inflamma-tion (nearly 60 % of BAL cells were eosinophils) whileIL-10 KO mice showed predominant ( G 90 %) infiltrationof mononuclear cells. Similar to the difference in propor-tions, the absolute number of eosinophils in BAL fluid ofIL-10 KO mice was significantly lower than that in wild-type control mice. Consistently, histopathological analy-

sis [Hematoxylin and eosin (H & E) staining] also showeddifferential patterns of respiratory tract inflammation inIL-10 KO and wild-type mice (Fig. 2). Wild-type miceexhibited diffuse and massive eosinophilic infiltration inalveolar, peribronchial and perivascular areas, whereasIL-10 KO mice showed significant mononuclear cell infil-tration with a tendency of more localized recruitmentaround peribronchial areas.

Mucus staining with thionin showed that OVA-sensitized/challenged IL-10 KO mice displayed signifi-cantly less goblet dell development in the bronchialmucosa compared with wild-type mice, indicating dimin-ished mucus secretion in the absence of endogenous IL-10 following allergen exposure (Fig. 3). The hyperplasiaof the bronchial epithelium in IL-10 KO mice was alsosignificantly less than that in wild-type mice followingallergen exposure. The marked reduction of goblet cellsand mucus production, together with the decrease ineosinophilic inflammation, indicates that the asthmaticreaction induced by allergen can be significantly blockeddue to the elimination of endogenous IL-10.

Eur. J. Immunol. 2000. 30: 382–391 Reduction of asthma-like reaction in IL-10 KO mice 383

Fig. 2. Cellular infiltration and distribution in the lung in IL-10 KO and wild-type mice shown by histological analysis. Mice weresensitized and challenged with OVA as described in Sect. 4.2. Lung tissues were routinely fixed, sectioned and stained withH & E. Sections were photographed at low (× 200, A, B) and high (× 400, C, D) magnification. (A, C) OVA-sensitized and -chal-lenged wild-type mice. (B, D) OVA-sensitized and -challenged IL-10 KO mice. A total of 12 mice in each group were analyzed andrepresentative findings are shown.

2.2 The reduction of asthma-like reaction iscorrelated with an alteration of cytokinepatterns in IL-10 KO mice

To explore the molecular basis for the altered cellularinfiltration into airways and mucus production in IL-10KO mice, we examined cytokine production patterns inthese mice following allergen sensitization/challenge.Antigen-driven IL-5 production by lymphocytes fromOVA-sensitized/challenged IL-10 KO mice was found tobe significantly lower than that in identically treated wild-type control mice (p X 0.01) (Fig. 4). In contrast, the pro-duction of Th1-related cytokines, IFN- + and IL-12, inIL-10 KO mice was significantly higher than that in wild-type mice (p X 0.001) (Fig. 4). Of note, IL-4 levels werenot significantly different between wild-type and IL-10KO mice (p = 0.26). For comparison, IL-5 productionstimulated by a polyclonal T cell activator (immobilizedanti-CD3 mAb) in IL-10 KO mice was comparable to that

of wild-type mice, although anti-CD3-stimulated IFN- +production in IL-10 KO mice was significantly higherthan that in wild-type mice (Fig. 5). Analysis of local cyto-kine production showed that IL-5 levels in BAL fluids fol-lowing OVA sensitization/challenge were significantlylower in IL-10 KO mice, compared with that in wild-typecontrols (Fig. 6). IL-4 and IFN- + were not measurable inBAL fluids at the time determined. The data suggest thatthe alteration in cytokine production, especially thedecrease in local and allergen-driven IL-5 responses,may be the basis for the altered cellular infiltration inIL-10 KO mice. The results also argue that IL-4 and IL-5,although both are Th2 cytokines, may be regulated dif-ferently by IL-10 in the antigen-specific response. Inaddition, the difference in IL-5 production of IL-10 KOmice following antigen-driven and polyclonal T cell acti-vation suggests that the antigen-driven cytokine patternis more representative of the in vivo profile of cytokineproduction induced by allergen exposure.


Fig. 3. Marked reduction of mucus production and gobletcell development within bronchial epithelium in IL-10 KOmice following OVA sensitization/challenge. Mice were sen-sitized and challenged with OVA as described in Sect. 4.2.Lung tissues were routinely fixed and sections were stainedas described in Sect. 4.3. Mucus was stained as purplishred or blue and cytoplasm was stained as orange color bythis method. All sections were photographed at the samemagnification (× 200). (A) Naive wild-type mice; (B) OVA-sensitized and -challenged wild-type mice; (C) OVA-sensitized and -challenged IL-10 KO mice. A total of 12 micein each group were examined and representative findingsare shown.

2.3 IL-10 KO mice show allergen-specificantibody responses comparable to those ofwild-type control mice

To elucidate the effect of the deficiency of endogenousIL-10 on antibody responses, we determined allergen-specific IgE, IgG1 and IgG2a responses in IL-10 KO andwild-type mice (Fig. 7). OVA-specific antibody responsesof all the three isotypes in IL-10 KO mice were compara-ble to those observed in wild-type mice. In particular, thelevels of OVA-specific IgE, an antibody isotype critical foratopic asthma, in IL-10 KO mice were virtually the sameas those in wild-type mice (Fig. 7 A). The results wereconsistent with the findings in the cytokine analysis thatshowed unchanged production, in IL-10 KO mice, of IL-4,a typical Th2 cytokine responsible for isotype switch ofIgE and IgG1 antibodies [24].

2.4 IL-5 production induced by OVAsensitization/challenge is dependent on CD4cells

The fact that IL-10 KO mice showed decreased IL-5 pro-duction implies that IL-10 might play a role in initiationand/or expansion of IL-5-producing Th2 CD4 cells.However, since IL-5 could be produced by many celltypes [25, 26], the question was whether CD4 T cellswere responsible for the observed allergen-driven IL-5production in this model. To address this question, weexamined IL-5 production by splenocytes from OVA-sensitized/challenged mice upon antigen-specific stimu-lation in the presence or absence of anti-CD4 mAb (YTS191.1). As shown in Fig. 8, in both wild-type and IL-10KO mice, OVA-driven IL-5 production by splenocyteswas virtually blocked ( G 80 %) by anti-CD4 mAb. Theresults indicate that CD4 T cells, in the OVA allergymodel, are the predominant cell type responsible for IL-5production. Since OVA-driven IL-4 production (Fig. 4)and IgE (Fig. 7) responses in IL-10 KO mice wee compa-rable to those in wild-type controls while OVA-driven IL-5production in IL-10 KO mice was significantly reducedcompared with wild-type mice, the data argue that theimmunoregulatory role played by IL-10 in allergicresponses may mainly target IL-5-producing Th2 cellsinduced by allergen exposure.

3 Discussion

The role of IL-10 in inhibition of Th1 cell development hasbeen clearly demonstrated in many model systems[27–31]. Although IL-10 is a cytokine mainly produced by


Fig. 4. Differential cellular recruitment into the airways iscorrelated with differential cytokine patterns in IL-10 KO andwild-type mice. Mice (four mice/group) were sensitized andchallenged with OVA as described in Sect. 4.2 and werekilled at day 7 post intranasal challenge. The splenocytesfrom individual mice were cultured at 7.5 × 106 cells/ml withstimulation of 1 mg/ml OVA. Culture supernatants were har-vested at 72 h and were tested for IL-5, IL-4, IFN- + and IL-12using ELISA. Data are presented as mean ± SD of eachgroup. ** p X 0.01, OVA-driven cytokine production in IL-10KO mice compared with that in wild-type mice. One repre-sentative experiment of three independent experiments isshown.

Fig. 5. Cytokine production by T lymphocytes in IL-10 KOand wild-type mice following polyclonal stimulation. Mice(four mice/group) were sensitized and challenged with OVAas described in Sect. 4.2 and were killed at day 7 post intra-nasal challenge. The splenocytes from individual mice werecultured in the presence ( ) or absence ( ß ) of immobi-lized anti-CD3. Culture supernatants were harvested at 72 hand tested for IL-5, IL-4 and IFN- + using ELISA. Data arepresented as mean + SD of each group. One representativeexperiment of three independent experiments is shown. **p X 0.01, IFN- + production in IL-10 KO mice compared withthat in wild-type mice.

Fig. 6. Decrease of IL-5 production in BAL fluids of OVA-sensitized IL-10 KO mice following allergen challenge inha-lation. BAL fluids from the mice described in Fig. 1 wereassayed for IL-5 levels using sandwich ELISA. Data are pre-sented as mean ± SD. * p X 0.05, IL-5 levels in IL-10 KOmice compared with those in wild-type mice.

Th2 cells in mice, its influence in the development of Th2cells remains unclear. The most significant finding in thisstudy is that IL-10 plays a promoting role in IL-5 produc-tion and bronchial eosinophilic reaction induced by aller-gen. Since IL-5 has been demonstrated to be critical ineosinophilic inflammation, the prevention of eosinophiliain IL-10 KO mice is most likely due to an inability of thesemice to mount IL-5 responses (Fig. 4). The findings inIL-10 KO mice are in contrast to the observations in stud-ies involving administration of exogenous IL-10, whichshowed that exogenous recombinant IL-10 inhibited invivo and in vitro IL-5 production by T cells and eosino-philic inflammation [15–18]. The reason remains unclearfor the discrepancy regarding the role of IL-10 in IL-5 andeosinophilic responses observed in previous studiesinvolving exogenous recombinant cytokine and thatshown in the present study using KO mice. The followingdifferences between the model systems used in previous

and the present studies, which may contribute to theobserved discrepancy, are obvious: (1) the study usingKO mice examines the function of endogenous IL-10,


Fig. 7. OVA-specific antibody production in OVA-sensitizedand -challenged IL-10 KO and wild-type mice. Mice weresensitized and challenged with OVA as described inSect. 4.2. Mice were bled at day 7 post intranasal challengewith OVA and serum OVA-specific antibodies in individualmice were determined. OVA-specific IgE was determined byPCA using female Sprague-Dawley rats. OVA-specific IgG1and IgG2a antibodies were determined by ELISA. PCA orELISA titers were transformed to log10 and presented asmean ± SEM. Pooled data from two independent experi-ments with similar results are shown.

Fig. 8. CD4 cells are responsible for IL-5 production in bothIL-10 KO and wild-type mice induced by OVA sensitization/challenge. Mice (four mice/group) were sensitized and chal-lenged with OVA as described in Sect. 4.2 and were killed atday 7 post intranasal challenge. The splenocytes from indi-vidual mice were cultured at 7.5 × 106 cells/ml with stimula-tion of 1 mg/ml OVA in the presence ( L ) or absence ( ß ) ofanti-CD4 mAb (YTS 191.1). Culture supernatants were har-vested at 72 h and tested for IL-5 using ELISA. Data are pre-sented as mean ± SD of each group. One representativeexperiment of three independent experiments with similarresults is shown. *** p X 0.001, IL-5 production in the wellswith anti-CD4 mAb compared with those without anti-CD4mAb.

therefore it is arguably more representative of the role ofIL-10 under physiological conditions, whereas the find-ings involving exogenously delivered recombinant IL-10may reflect the effect mediated by excessive, possiblynon-physiological, levels of IL-10; and (2) administrationof exogenous IL-10 mainly tests the immediate effect ofthis cytokine on cell development and allergicresponses, whereas studies using KO mice examine theeffect of chronic IL-10 deficiency on allergic reaction.Moreover, it is also possible that IL-10 plays oppositeroles in the induction phase versus effector phase ofasthmatic reaction. Specifically, IL-10 may play a pro-moting role in the induction phase of eosinophilicresponses by enhancing IL-5 production, while playingan inhibitory role in established eosinophilic inflamma-tion via down-regulating IL-5 production by polarizedTh2 cells and by direct effects on eosinophils.

One caveat when using IL-10 KO mice might be thatthese mice have been reported to spontaneouslydevelop Th1-associated inflammatory bowel disease at3–4 weeks of age [32], thus possibly interfering non-specifically with the process of allergic reaction. TheIL-10 KO mice on C57BL/6 background used in thepresent study which were obtained from Jackson Labo-ratories do not normally develop inflammatory bowel dis-ease when they are kept in a “clean” environment [33,34]. The IL-10 KO mice used in the present study werealways kept in an SPF facility and the cages, food andwater were all autoclaved. All the IL-10 KO mice used inthe study showed body weight comparable with wild-type controls during the study period. Moreover, sixOVA-immunized IL-10 KO mice were examined for intes-tinal pathology and none of them showed changes char-acteristic of inflammatory bowel diseases. Therefore, theinfluence, if any, of spontaneous inflammatory bowel dis-ease on the present model system of allergic response isminimal.

Interestingly, alterations in IL-5/eosinophilia responsesand mucus production observed in IL-10 KO miceappear to be disassociated from IL-4/IgE responses inthis model system. Although IL-10 KO mice exhibiteddecreased IL-5 and pulmonary eosinophilic responsesand bronchial mucus production in comparison withwild-type control mice, IL-4 and IgE production amongthese two groups of mice were comparable followingOVA sensitization/challenge. The results suggest that,albeit bronchial eosinophilia, mucus production, IgEresponses and IL-4/IL-5 production often co-exist dur-ing allergic responses such as asthma, the regulatingmechanism for these various components may operateindependently. Accordingly, a factor which directly orindirectly induces IL-5/eosinophilic reaction and mucusproduction may not show significant impact on IL-4/IgE


production. Indeed, clinical studies have shown thatsome patients exhibit improved clinical status after aller-gen immunotherapy without measurable changes inallergen-specific IgE responses [35, 36]. IL-10, therefore,may be a critical factor for the initiation/expansion ofIL-5-producing Th2 CD4 cells without significant in-fluence on IL-4-producing cells. Moreover, since theIL-4/IFN- + ratio has been demonstrated to be importantin regulating IgE and IgG2a responses, it is surprising thatthe IL-10 KO mice which exhibited a remarkable increaseof IFN- + , thus a significantly altered IL-4/IFN- + ratio, didnot show significant change in IgE and IgG2a production.Although the reason for this phenomenon was not identi-fied in this study, the results suggest that antibody isoty-pes may be regulated by multiple mechanisms.

The coincidental alteration of reduced IL-5 and elevatedIFN- + production in IL-10 KO mice following allergenexposure suggests that the reduction of IL-5 may be par-tially caused by the increase of IFN- + which inhibits IL-5-producing Th2 cells. IFN- + has been shown to be inhibi-tory for Th2 responses [37–39]. It was also reported thatsystemic [40] and bronchopulmonary [41] delivery ofrecombinant IFN- + to OVA-sensitized/challenged miceprevented allergic eosinophilia into airways. Studies ininfectious disease models using KO mice also suggestan inhibitory role of IFN- + on IL-5 production. For exam-ple, IFN- + KO mice show increased Th2 cytokine pro-duction, including IL-5 and IL-10, when immunized witha mycobacterial protein (PPD) which induces Th1 cyto-kine production in wild-type control mice [42]. We alsofound that IL-5 production by CD4 cells was significantlyreduced in IL-10 KO mice which showed significantlyelevated IFN- + production following infection with Chla-mydia trachomatis, an intracellular bacterium [34]. On theother hand, since IFN- + is likely to inhibit both IL-5 andIL-4/IgE production, the rather selective reduction ofIL-5, but not IL-4/IgE production in IL-10 KO mice in thepresent study suggest that inhibition of IFN- + productionmay not be the sole mechanism by which IL-10 pro-motes IL-5 and asthmatic reaction.

Finally, although eosinophil infiltration was significantlyinhibited in IL-10 KO mice, remarkable pulmonaryinflammatory cellular recruitment was observed in thesemice following OVA sensitization/challenge. This findingindicates that the same antigen in different cytokinemicroenvironments may induce different patterns ofinflammatory cellular infiltration. Moreover, it indicatesthat, although allergic eosinophilia and mucus produc-tion can be prevented/treated by approaches whichinhibit or delete IL-10 in vivo, remarkable mononuclearcell – including lymphocyte – inflammation still existsamong IL-10-deficient mice following allergen exposure.The latter point may be particularly relevant to immuno-

regulation of atopic asthma because there are reportsshowing that lymphocytes, in the absence of eosino-phils, may confer end-stage events in allergic airwayinflammation, such as airway hyperreactivity in somemouse and rat allergy models [43–47]. Since airwayhyperreactivity was not examined in this study, the corre-lation between reduced eosinophilia/enhanced mononu-cleated cell infiltration and asthmatic reaction is notclear. Further studies, to elucidate the significance ofthese non – (or less) eosinophilic inflammation, espe-cially its effect on bronchial hyperreactivity, are requiredbefore therapeutic approaches to allergic diseases aim-ing at manipulating IL-10 synthesis can be considered.

On the other hand, the dramatic decrease of bronchialmucus production and mucosal hyperplasia (Fig. 3) inIL-10 KO mice following OVA exposure suggest a clinicalbenefit for asthma due to the deficiency of endogenousIL-10. Substantial increase in mucus secretion by airwayepithelium is an important factor in asthmatic reaction. Inparticular, mucus hypersecretion has been found to behighly associated with asthma-induced death. Therefore,blockade of mucus hypersecretion is closely relevant tothe treatment and prevention of asthmatic reaction. Themechanism by which IL-10 regulates goblet cell devel-opment and mucus production is not clear. Since IL-4and IL-13 have been found to be the crucial cytokines inregulating mucus production and goblet cell develop-ment [48–50] and since no significant alteration in IL-4production was found in IL-10 KO mice (Fig. 4), it is likelythat IL-13 production in these mice was altered due todeficiency of endogenous IL-10. Studies to confirm thishypothesis are currently ongoing.

4 Materials and methods

4.1 Animals

Female homozygous IL-10 KO mice (IL-10–/–) (C57BL/6-IL10 <tm1Cgn>) were purchased from the Jackson Labora-tories (Bar Harbor, ME). Age- and sex-matched wild-typeC57BL/6 mice were purchased from Charles River Canada(St. Constant, PQ, Canada). Female Sprague-Dawley ratswere bred at the University of Manitoba (Winnipeg, Canada)breeding facility. Animals were used in accordance with theguidelines issued by Canadian Council on Animal Care.

4.2 Immunization and BAL

Mice were initially sensitized i.p. with 2 ? g OVA (ICNBiomedicals, Montreal, Canada) in 2 mg Al(OH)3 adjuvant(alum). At day 15 post sensitization, mice were challengedintranasally with 100 ? g OVA(40 ? l) and were killed at various(2–10) days following the challenge. The trachea were can-


nulated and the lungs were washed twice with 1 ml PBS.The BAL fluid was centrifuged immediately and cells wereresuspended in 0.5 ml PBS. Total BAL cells were firstcounted and BAL smears were then prepared for differentialcell counting. The slides were air dried, fixed with ethanoland stained with Fischer Leukostat Stain Kit (Fishers scien-tific, Ontario, Canada) to stain leukocytes. The number ofmonocytes, neutrophils, lymphocytes and eosinophils in200 cells were counted based on morphology and stainingcharacteristics.

4.3 Histopathological analysis

Lung tissues were fixed in 10 % buffered formalin, embed-ded in paraffin, sectioned, stained with H & E and examinedfor pathological changes under light microscopy. Mucus andmucus-containing goblet cells within bronchial epitheliumwere analyzed by thionin staining using the Mallory method[51] and counterstaining with orange G.

4.4 Cytokine analysis

For examination of cytokine production patterns in spleno-cytes, mice were killed at day 7 following intranasal chal-lenge with OVA and spleen cells were cultured in vitro aspreviously described [52]. Briefly, cells were cultured at 7.5 ×106 cells/ml (2 ml/well) alone or with immobilized anti-CD3mAb (145-2C11), purchased from PharMingen (San Diego,CA) with 1 mg/ml OVA in the presence or absence of anti-CD4 mAb (YTS 191.1) at 5 ? g/ml. Duplicate cultures wereestablished from the spleen cells of individual mice in eachgroup. Culture supernatants were harvested at 72 h formeasurement of different cytokines using ELISA. Purified(for capture) and biotinylated (for detection) antibodies pur-chased from PharMingen were used for the ELISA as previ-ously described [34]. IFN- + levels were tested by a two mAbsandwich ELISA (XMG1.2 for capture and R4-6A2 for detec-tion). IL-5 levels were tested using mAb TRFK as captureantibody and mAb TRFK4 as detection antibody. IL-4 levelswere measured using 11B11 as capture antibody and BVD6-24G2 as detection antibody. IL-12 (p40) levels were meas-ured using mAb C15.6 as capture and mAb C17.8 as detec-tion antibody, respectively. Cytokines in BAL fluids were alsodetermined by these ELISA.

4.5 Antibody analysis

OVA-specific IgE was determined by passive cutaneousanaphylaxis (PCA) of Sprague-Dawley rats as described[53]. Allergen-specific IgG1 and IgG2a were measured byELISA using goat anti-mouse IgG1 or goat anti-mouseIgG2a antibodies purchased from Southern BiotechnologyAssoc. Inc. (Birmingham, AL) as described [53].

4.6 Statistical analysis

Antibody titers (ELISA or PCA) were log 10 transformed andanalyzed by unpaired Student’s t-test. Cytokine levels anddifferential BAL cell counts in different groups were analyzedby unpaired Student’s t-test.

Acknowledgements: This work was supported by agrant from the Medical Research Council of Canada (MRC)to X.Y.(MT-14680). X.Y. holds a salary (scholar) award fromMRC.

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Correspondence: Xi Yang, Department of Medical Microbi-ology, Faculty of Medicine, University of Manitoba, Room523, 730 William Avenue, Winnipeg, Manitoba, Canada R3EOW3Fax: +1-204-789-3926e-mail: yangxi — cc.umanitoba.ca


il-10 deficiency prevents il-5 overproduction and eosinophilic inflammation in a murine model of...

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