Iraqi Journal Of Pharmaceutical Sciences Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Evaluation of the role of IL-2 and IL-4 in asthma 47 Evaluation of the Role of Interleukein-2 and Interleukein-4 in the Immunopathogenesis of Steroid Therapy Resistance in Iraqi Asthmatic Patients Adel A. Sahib * , 1 , Abdul Wahab A.Al-Shaikely ** * AL-Nassyria General Hospital **Department of Clinical Laboratory Sciences,College of Pharmacy,University of Baghdad,Baghdad ,Iraq Abstract Interleukins (IL-2 and IL-4) are increased in asthmatics and were reported to induce resistance to steroid therapy in some patients who fail to get benefit from glucocorticoids when used in full dose and for long period of time. In this context, the present study was conducted on Iraqi patients to provide additional laboratory mean, beside the clinical diagnosis, for the decision whether the asthma is steroid sensitive or resistant by monitoring the level of immunoglobulins, complement proteins and interleukins among asthmatic patients (steroid sensitive or resistant) and the possible contribution of other factors like age, sex and environments in the development of steroid resistance. A total number of 55 asthmatics and 28 normal subjects were enrolled in the study. Patients were diagnosed clinically as steroid sensitive (SSA) and steroid resistant (SRA) and blood samples were taken from all subjects included in the study for the measurement of immunoglobulins (IgA, IgG, IgM and IgE), complement proteins (C3 and C4), interleukins (Il-2 and Il-4), and total and differential WBC counts.The results showed no age, sex and residence dependency of acquired steroid resistance, while smoking habit (and may be the atopic allergy) constitute marked predisposing factors. The level of IgA and IgE were high in both SRA and SSA, while IgG level was low in SRA. Complement proteins (C3 and C4) were not differ in asthmatic patients in comparison with control group. The interesting results were those concerning interleukins. The levels of IL-2 and IL-4 were very high in SRA than in SSA. These are parallel with high lymphocyte and neutrophil counts in blood samples of those patients.In conclusion, beside clinical diagnostic features concerning the dose and duration of therapy with glucocorticoids, monitoring the levels of IL-2 and IL-4 could provide additional laboratory diagnostic measures for the convincing decision that asthma is steroid resistant. Key words: steroid resistant asthma, steroid sensitive asthma, IL-2, IL-4. ةصالخلا ة فسفي مظا ي لحيسىا ضعريسوىظلض كديزسىوقضدكديدقعي تخليسىت ايزنعيةياايمىاي ييضيىتلالي لحيسىلا ضعيسىوتسم ديدزي ية فسفيدقعي تخليسىت ايزنعيinterleukin-2 and interleukin-4سىولك ديىهوتج .زنعيىااليداي مظا يسوحظتىكاوكقضعيز ي كقلضي ةصقظاي ضز ديخعيسىلاسي ضىمظكتزةعسعيدقعي لحيسىوتخليسى ة يسظلتزسيما يسو ظ ض ديىهمظكتزةعسعيدقعيس ظاعس لضي تدلضيسى ثا ي .زملي سيسىمكضليم عيدصاتةييسىعلس ديسىفضىكدي خضمديز كهدي اظيتةدي ض خضمديلىليسىظااكةيسىمتةتديىهظوي ي يسىظ تةتيةزىوظتةيزاة يسىاها كقضعيسىوقضدكد تسنميدي مظا مكوضيلمسيوضحيياضىديسىت اي مظ كيديدزي ضز ديىهلاسي ضىمظكتزةعسعيزمىاي ياايي (imunoglobulins)ىددتيتسي تزتك يسي ي (complement proteins)يز(سوحظتىكاوكقضعيinterleukins)ي ك ي تخليسىت اي يمليعكد لسىلاس يسياقسيزسيدوتيزسييسىومض وديسىوفظوهديىهلاس ليسةات ي ل يي ض خضمديىعلس د(يىهمظكتزةعسعيدمزي ضز اا مظ كياا) ي يسةتفضريملي مظاوليسىقضتتةديسىلضتيزىهوظتةي يايضذي28ي تةدضاي ضىت ايز55دصاتةييسىعلس ديدهلي .ىهمظكتزةعسع ضز ديسيتساةتي .تتيسا ي كضحضعيوض هديد يسىوتخليزسى ة يوضحاسينعيااثاسي تةتةضايوو ضز ك يدزي مظ كيك يىهلاسي2005يلىليتاتة يدزيي2005 ,IgA, IgG ضىمظكتزةعسع .زتتيسا يدكقضعيسىعتي يو يسةااضبيسىواضلوك ي ضىعلس ديزمىايى كض ي مظا يسىاها كقضعيسىوقضدكدي) IgM, IgEيزسىيتزتكقضعيسىظيوكهكد ) (C3يز C4(يزسوحظتىكاوكقضعي )IL-2يزيIL-4ض خضمديى كض يدعفيوتةضعيسىعتيسىيكدضري ) داي ضز ديسىلاسي ضىمظكتزةعسعيركتي لظوعةيدهليسىلوتيدزي ةيسىعلس دليدظلتعيحظضعاي .(Differential countزلاثضعلضيسىظوضخهلي) كلِديتداس ميك يتضاكتسيزسخفض(يسىووتزدىفمض كدمزل وضييفاك ي)وضايىهيي ي كقوضيي(ةيورق مأ ةيرضح تناك نإ) لايوضحييادتةديدتينتزةد(يسىمي سى قسيدزي قس د ي كقوضية يزسىومظ كيك يىهمظكتزةعسع(سىو ضزت) سىثقوك ي ي تخليسىت ايوايدقع ةضايوضايدضيIgEيزIgA مظا يلاي .ىفعزقيسىو ضز د ( ركتيC4 يزC3زنعيزاعيدةدضاي ضاي مظا يسىيتزتكقضعيسىظيوكهكدي) .يىهمظكتزةعسع وادديسىو ضز دسي دقع ساي قاوحIgGوضاي مظا ي اظه يدقعيوايسىثقوك ي يسىوتخلي ضىو ضلحدي ي وادديسةااضبيسةتفضر . يسىقظضعايسىولكتةيملي ةيسىعلس ديتهايسىوظله دي ( زسىظليوضحيي مظاةضتلضيدضىكدياعسايدقعيسىو وادديسىو ضز ديىهمظكتزةعسع .ز ةيسىقظضعاياضرعي ظاساةديIL-4 يزIL-2 ضوحظتىكاوكقضعي) ةوي يسو ظقظضسي ي ةيسىعلس ديسحجي ض خضمديلىلي يسىهووضزةديزسىولظعىديدقعيدزىِايسىوتخل.ر يسىلعفيسىوتتو يىيتةضعيسىعتيسىيكدض يزIL-2سىظااكةيسىمتةتديمكوضيةظله ي ضى تدديز عةيسىلاسي ضىمظكتزةعسعيىوتخليسىت ايمضاي تسنيدي مظا يتتوك يسوحظتىكاوكقضعي) IL-4يسىقاايسى ديةمظ كييدزية ضزتيسىلاسيا( ةوي يدايةدك يز كهدي اظيتةديىهومضدعةيمليت تةتيمكوضيلمسيوضحيياضىديسىت ايت ضىمظكتزةعسع. 1 Corresponding author : E-mail : adph70@yahoo.com Received : 14/5/2007 Accepted : 24 /6/2008 mailto:adph_70@yahoo.com Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Evaluation of the role of IL-2 and IL-4 in asthma 48 Introduction Increased responsiveness to a variety of stimuli is a common feature involved in the one of the interesting lung diseases, asthma (1). Whether hypersensitivity to inhaled allergens is present or not, asthma is categorized broadly into extrinsic and intrinsic types, respectively(2,3). The incidence of extrinsic asthma occurs most frequently between the age of 3 and 45 years, although its onset may be at any age (4). Serum immunoglobulin E (IgE) antibody level is raised in most of those patients (5). Intrinsic asthma, on the other hand, has a primary onset before the age of 3 years, or after 35 to 45 with, however, some hypersensitivity to drugs, particularly aspirin and some other manifestations including nasal polyposis and urticaria (6). Other category of asthma are causally related to different inducing factors including occupational, exercise-induced and aspirin-induced asthma(7,8). The role of immunity and related inflammatory cytokines in the pathogenesis of asthma has been extensively studied. Interleukin-2 (IL-2) is a known T-cell growth factor which induces clonal expansion of T- lymphocytes (9). Activated T-cells, B-cells and mast cells have the ability to synthesise IL-2 (10,11). The second important T-cell growth factor is interleukin-4 (IL-4), which is synthesized by some types of T-lymphocytes, basophils, eosinophils and mast cells to function as mitogenic and lymphocyte differentiation factor(11). These cytokines have, beside these functions, anti-tumor effects on some types of tumors(12,13). In addition to activation of immune system during the pathogenesis of asthma, neuropeptides (substance P and neurokinine A) and nitric oxide have shown to play major roles in the initiation of cascade of responses including vasodilation, mucous secretion, plasma protein extravasation, leukocyte adhesion and activation, and bronchoconstriction that comprise the classical signs and symptoms of asthma (14). These pathogenic mechanisms are controlled to some extent by the use of glucocorticoids (GC) (15). However, some resistance to this mode of therapy is continuously increased and is manifested by failure to improve baseline morning (AM) pre- bronchodilator forced expiratory volume during the first second (FEV1) by greater than 15% following 7-14 days of 20mg twice daily oral prednisolone (16). Some patients do not have an absolute resistance, but rather GC insensitivity and some might respond to higher doses of prednisolone or require longer period of therapy (17). Patients with steroid resistant asthma (SRA) have higher level of immune activation (raised levels of T-cells and eosinophils with high level of IL-2 and IL4)(18). This accompany by persistent respiratory symptoms, nocturnal exacerbations and chronic airway obstruction together with poor clinical and physiologic responses to oral glucocorticoids (GCs) therapy (17). In clinical practice, setting any patient not responding to 40-60mg/day prednisolone after 3 weeks of therapy should be suspected to be SRA (16). The clinical efficacy of GCs therapy is the result of the combinations of inhibitory effects on the process of inflammation including decreased trafficking of inflammatory cells and inhibition of inflammatory cytokines production (19). This efficacy has shown to be altered in SRA (20). The correlation between increased inflammatory cytokines (IL-2 and IL-4) and the development of steroidal resistance have been studied. Cytokines were reported to induce activation of transcriptional factors that interfere with GC binding to their nuclear recognition sites (21,22). IL-2 and IL-4 were shown to promote the synthesis of altered GC binding protein (GCR), which reported to be a dominant negative inhibitor of the classic ligandيbindingيproteinيforيGCي(GCRα (ةيورق مأ ةيرضح تناك نإ)(23,24,25)ي. Factors contributing to GC insensitivity via immune activation may include allergen exposure which is reported to decrease GC receptor binding affinity and steroid responsiveness in atopic asthmatics (26) possibly by IL-2 and IL-4 dependent mechanisms(17). On the other hand, superantigen secretion by bacterial or viral agents my contributed to poorly controlled asthma and reduce GC sensitivity. In this context, staphylococcal enterotoxin B is a potent inducer of GCR isoform in T-cells (27). According to previously mentioned immunopathogenic features of steroid resistant asthma, this study was conducted to monitor the role of IL-2 and IL-4 in the immunopathogenesis of steroidal resistance asthma among Iraqi patients; and to monitor any role for immunoglobulins, complement proteins, age, sex, residence and smoking habit on the incidence of SRA. Patients, Materials and Methods This study was conducted on 55 asthmatic patients (25 females and 30 males) and another 28 healthy persons (18 females and 20 males) in a single-blind technique. The study was carried out in AL-Nasseriya General Hospital from February 2005 till October 2005. The age range of healthy subjects and patients was 16-73 years with average age ± SD (39.4 ± 14.67). Steroid sensitive asthmatics (SSA) were 28 (10 females and 18 males) and Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Evaluation of the role of IL-2 and IL-4 in asthma 49 SRA were 27 (12 female and 15 males). Patients' selection was based on special criteria including (a) presence of no acute infection at the time of study, (b) presence of no any other chronic infection, (c) steroidal therapy must be discontinued at least for 2 weeks and (d) presence of no systemic disease that my be associated with steroid resistance. Patients were diagnosed for steroidal resistance depending on the history of steroidal therapy and the clinical decision. Other patient's information were collected in a specially prepared sheet including: age, sex, chief complain, type and dose of steroid used, predisposing factors, associated symptoms, medical history, family history of steroid resistance, smoking habit, residence (civilian or rural), and presence of atopic allergy. Materials: IL-2 and IL-4 Elisa Kit (Immunotech, Marseille, France), Single Radial Immuno- diffusion Test Kit for immunoglobulins (BINDARIDTM The Binding Site Ltd., Birmingham, UK), Single Radial Immunodiffusion Test Kit for Complements (BINDARIDTM The Binding Site Ltd., Birmingham, UK), IgE Elisa Kit (Immunotech, Marseille, France). Methods: Blood samples were drawn, left for clotting and then centrifuged for 5-10 minutes at 2000 rpm (using Centrifuge, K24, Coold With Rotor Number 905, WIR 12x10 ML, Janetzki, Germany) for separation of serum, which was kept frozen unless analyzed immediately. Serum levels of IL-2 and IL-4 were determined using ELISA kits (Immunotech, Marseille, France), based on the interaction between monoclonal antibody bound to the wells of a microtiter plate to the IL-2 and IL-4 found in the serum. The antigen- antibody complex was detected by the addition of a chromogenic substrate, and the intensity of color was recorded colometrically (using Spectrophotometer SP6-500, Pye-Unicam, England); accordingly serum levels of IL-2 and IL-4 were calculated utilizing a standard curve prepared for this purpose (28). Serum level of the Immunoglobulins (IgG, IgA, and IgM) and the complement proteins (C3 and C4) were determined using SRID kits (BINDARIDTM The Binding Site Ltd., Birmingham, UK). Equal volumes of reference sera and test samples were added to wells in an agarose gel containing a monospecific antiserum. The samples diffused radially through this gel and the tested compound (antigen) being assayed by forming a precipitin ring with the monospecific antiserum; rings diameters were measured (using Microwell System Reader 2305, Organon Teknika, Austria), and concentrations were determined using standard curve prepared for this purpose(29). IgE was determined using IgE ELISA kit (Immunotech, Marseille, France) Statistical Analysis All results were presented as a mean  SEM. Comparisons were made using Chi- square,يStudent’sيt-test and ANOVA. P values less than 0.05 were considered significant. Results In this study, age distribution of the volunteers enrolled in the study revealed that the incidence of SRA was varied but generally is great in the middle and older ages (Table 1). Table (1): Distribution of patients according to their age among steroid resistant and sensitive asthmatic patients. Age (years) SRA (%) (n=27) (32.5%) of total (83) patients SSA (%) (n=28) (33.73%) of total (83) patients 10-20 --- 3.57 21-30 11.11 17.85 31-40 33.33 28.57 41-50 11.11 21.42 51-60 11.11 14.28 > 61 33.33 14.28 SRA = Steroid resistant asthma. SSA = Steroid sensitive asthma However, no significant difference (P>0.1) was noticed in the incidence of SRA among female (55.56%) and male (44.44%) with male/female ratio of 0.33 (Table 2). On the other hand, the incidence of SRA was shown to be high in those with negative family history (77.77%) in comparison to those with positive family history (22.23%) (P<0.01) as shown in table (2). In addition, steroid resistance was more pronounced in civilian (66.67%) than in rural areas (33.33%); but however the difference was not significant (P>0.1) (Table 2). It is clearly shown that smoking may comprise significant predisposing factor for steroid resistance (55.56%) when smoking SRA patients were compared to non-smoking Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Evaluation of the role of IL-2 and IL-4 in asthma 50 SRA patients (44.44%) (Table 3). Atopic allergy, on the other was more pronounced in patients with SRA (66.66%) in comparison to SRA patients with no symptoms of atopic allergy (34.34%); however, the difference was not significant, P>0.5 (Table 3). Table (2): Sex distribution, family history and residence among SRA and SSA patients included in the study. Asthm- atic groups Sex Family history Residence Male (%) Female (%) Positive (%) Negative (%) Civilian (%) Rural (%) SSA (n=28) 64.28 35.72 71.42 28.57 82.14 17.85 SRA (n=27) 44.45 55.55 22.22 77.78 66.67 33.33 P-values >0.1 <0.01 >0.1 Table (3): Smoking habit and atopic allergy among steroid resistant and steroid sensitive asthmatic patients. Asthmatic groups Smoking Atopic allergy Non- smokers (%) Smokers (%) Present (%) Absent (%) SSA (n=28) 82.15 17.85 71.43 28.57 SRA (n=27) 44.45 55.55 66.66 33.34 P-values <0.05 >0.5 SRA group of patients exhibited varying patterns of immunoglobulin levels as shown in figure (1). There is significant elevation in IgA level (3105.55 ± 225.12) and IgE level (295.17 ± 61.5) in comparison to control group (2346.11 ± 142.7 and 39.05 ± 4.5, respectively) (P<0.05). However, the levels of these immunoglobulins also were significantly high in SSA group (3815.71 ± 302.61 and 387.85 ± 52.5, respectively) (P<0.05). Moreover, only IgA levels were shown to be significantly different between SRA and SSA. The level of IgG level did not differ significantly in SRA (13615.56 ± 993.23) over that in control group (12798.33 ± 746.27) (P>0.05); but it was significantly high in SSA (15890.36 ± 892.08) (P<0.05). Lastly, the level of IgM did not differ significantly among control (1626.89 ± 139.6), SRA (1407.11 ± 187.9) and SSA (1593.82 ± 136.38) groups (P>0.05) as shown in figure (1). 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 IgA IgG IgE IgM Im m un og lo bu lin le ve l ( m g/ L) Control SRA SSA a a a a a b a b c a b b Fig. (1): Immunoglobulins level among SSA (n=28) and SRA (n=27) patients. Values are (mean ± SEM). Non-identical superscripts (a, b, c) considered significant, P<0.05 analyzed by ANOVA. In this study also, the levels of complement protein (C3) did not differ significantly in SRA (1834.44 ± 46.48) and SSA (1666.78 ± 58.21) in comparison with control group (1699.44 ± 46.48), P>0.05 (Fig. 2). The same profile was seen in the second complement protein (C4) who its level show comparable values in SRA (398.44 ± 20.3) and SSA (389.82 ± 25.5) to that in control group (375.72 ± 23.5), P>0.05 as shown in figure (2). 0 200 400 600 800 1000 1200 1400 1600 1800 2000 C3 C4 C om pl em en t p ro te in le ve l ( m g/ L) Control SRA SSA a a aa aa Fig. (2): Complement proteins level among SSA (n=28) and SRA (n=27) patients. Values are (mean ± SEM). No significant difference among groups, P>0.05 analyzed by ANOVA. The interesting results in this study were those related to cytokines (IL-2 and IL-4) levels. Both cytokines were significantly high in SRA (46.11 ± 2.31) and SSA (18.28 ± 0.398) in comparison to control group (8.38 ± 0.63), P<0.05; however, its level among SRA was significantly higher than that in SSA group (P<0.05) as shown in figure (3). Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Evaluation of the role of IL-2 and IL-4 in asthma 51 0 10 20 30 40 50 60 70 80 90 100 IL-2 IL-4 In te rl eu ki n le ve l ( p g/ L) Control SRA SSA cb c a b a Fig. (3): Interleukins level among SSA (n=28) and SRA (n=27) patients. Values are (mean ± SEM). Non-identical superscripts (a, b, c) considered significant, P<0.05 analyzed by ANOVA. On the other hand, the same profile was seen for IL-4 who its level was significantly high in SRA (91.33 ± 4.42) and SSA (49.03 ± 2.13) in comparison to control group (26.05 ± 11), P>0.5; with significant difference among the two groups of asthmatics, P<0.05 as shown in figure (3). Total WBC counts were significantly high in SRA and SSA in comparison to control group, P<0.05 (P<0.05) as shown in figure (4). 0 1 2 3 4 5 6 7 8 9 10 Cont rol SRA SSA T ot al W B C c ou nt (x 10 00 /d L ) a b b Fig. (4): Total WBC count among SSA (n=28) and SRA (n=27) patients. Values are (mean ± SEM). Non-identical superscripts (a, b) considered significant, P<0.05 analyzed by ANOVA. Further, the count did not differ significantly among SRA and SSA groups (P>0.05). Neutrophils were significantly high in SRA and SSA groups in comparison to control group (P<0.05). However, their level did not differ significantly among SRA and SSA as shown in figure (5), P>0.05. Lymphocyte levels were low in both groups of asthmatics using steroidal therapy in companion to control group; however it was slightly lower in SSA than in SRA, but the difference was not significant (P>0.05). Differential count of other WBCs did not show any significant difference in SRA and SSA in comparison to control group (P>0.05) as shown in figure (5). 0 10 20 30 40 50 60 70 80 Basoph. Eosinoph. Mono. Lympho. Neut roph. D if fe re nt ia l C ou nt ( % ) Cont rol SRA SSA a b b a b b a a aa a a Fig. (5): Differential WBC count among SSA (n=28) and SRA (n=27) patients. Values are (mean ± SEM). Non-identical superscripts (a, b) considered significant, P<0.05 analyzed by ANOVA. Discussion The present study showed that the incidence of steroid resistance is increased among all patients' age rang. This is come in agreement with that reported by Ishioka and his co-workers(30). However, resistance to steroid did not significantly affected by sex variation, but may be linked to other factors such as smoking habit and presence of atopic allergy, a finding supported by what reported by others (31,32). Atopic allergy and immune activation is clearly suggested in this study to underlay steroid resistance and this is supported further by finding in this study that the levels of IgA and IgE were high in those patients (33). Furthermore, the low or un increase level of IgG in SRA in comparison to SSA group suggest that the decrease in this immunoglobulin to be one factor contributing to steroid insensitivity. This has been solidified by the question; why Nathan and Erwin introduced IgG as intravenous immunoglobulin in the treatment of steroid resistance; this in turn based on the ability of IgG to decrease the levels of IL-2 and IL-4 in vitro, an effect thought to be involved in the potentiation of inhibitory effect of glucocorticoids on cell proliferation and cytokine secretion(34).The link between the high concentrations of IL-2 and IL-4 and the development of SRA relay on the increased resistant of lymphocytes to the action of GCs in a theory suggest altered splicing of the GCR Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Evaluation of the role of IL-2 and IL-4 in asthma 52 pre-mRNA genes induced by these cytokines (35). The results is the generation of a second GCR,يtermedيGCRβ,يwhichيdoesيnotيbindيGCي but antagonizes the transactivating activity of the classic GCR (25). Thus, increased expressionيofيGCRβيcouldيaccountيforي glucocorticoid insensitivity among asthmatic patients (36,37). For this reason, the use of high dose of glucocorticoids might make down regulation to the classical glucocoricoid receptors (GCRα (ةيورق مأ ةيرضح تناك نإ),يleavingيtheيinhibitoryي isoformي(GCRβ (ةيورق مأ ةيرضح تناك نإ)يtoيbeيpredominate,يandيthatيisي why resistance occurs to GCs (23). The levels of C3 and C4 did not differ significantly in this study among SRA and SSA in comparison with baseline. For this reason we suppose monitoring the level of these complements is without benefit to decide wither the patient has steroid sensitivity of resistance. This speculation was come in agreement with that reported by Liao and his associates (38). The elevated levels of IL-2 and IL-4 seen in this study are correlated well with the acquired resistance to steroid therapy. Positive correlation was existed between the increased levels of IL-2 and IL-4 among SRA patients although the correlation failed to reach the level of statistical significance (data not shown). These results came in agreements with those reported by Kam and his co-workers (1993) in that the combination of IL-2 and IL- 4 induced T cell resistance to GCs and increase GCR expression in the T cells of normal subjects (39). In mice IL-2 alone can induce T cell resistance to GC (40). The mechanism of such resistance involves a defect in nuclear translocation of the GC receptors. This in turn depends upon the phosphorylation of GC receptors (41). Thus, the results obtained in this donate a possible usefulness of IL-2 and IL-4 as predictive immune markers for the development of steroid resistance and to be a possible underling cause for such resistance (23, 42). The levels of total WBC were increased in both groups of asthmatic patients. Further, the percentage of lymphocytes and neutrophils were high in SRA group in comparison to SSA group, although the change was not significant. The high level of these leukocytes in SRA based on the theory that glucocorticoids intake could inhibit cell proliferation because of the high dose of steroid used in asthmatic patients as what happen in SSA patients (43).In conclusion, beside clinical diagnostic features concerning the dose and duration of therapy with glucocorticoids, monitoring the levels of IL-2 and IL-4 could provide additional laboratory diagnostic measures for the convincing decision that asthma is a steroid resistant. Acknowledgment The authors gratefully thank the College of Pharmacy, University of Baghdad for supporting this research and pharmacist Haider M. Mohammed for his assistance in preparing the manuscript . References 1. National Institutes of Health (NIH): Guidelines for the Diagnosis and Management of Asthma. National Asthma Education Program Expert Panel Report, DHHS publication 1991; No. 91- 3042. 2. Humert, M.; Menz, G. and Ying, S.: The immunopathology of extrinsic (atopic) and intrinsic (non atopic) asthma: more similarities than differences. Immunol. Today 1999; 20: 528-533. 3. Varney, V.A. and Holgate, S.T.: Allergy testing in respiratory medicine. Brit. J. Hosp. Med. 1996; 56: 406-408. 4. Van Arsedel, P.P. and Larson, E.B.: Diagnostic tests for patients with suspected allergic disease. Ann. Intern. Med. 1989; 110: 304-312. 5. Burrows, B.; Martinez, F.D. and Halonen, M.: Association of asthma with serum IgE levels and skin test reactivity to allergens. N. Engel. J. Med. 1989; 320: 271-277. 6. Buss, W.W. and Reed, C.E.: Asthma definition and pathogeneses. In: Middleton, E.jr. Reed; C.E.; Ellis, E.F., (Eds.). Allergy: principle and practice (3rd ed.). C.V. Mosby, St. Louis, 1988; PP: 969. 7. Anderson, D.W. and Rosenberg, M.: Asthma: General concepts. In: Patterson, R., (Ed.). Allergic disease: Diagnosis and Management (3rd ed). Lippincott Company, Philadelphia, 1985; PP. 252- 301. 8. Edwards, C.R.W.; Bouchier, I.A.D. and Haslett, C.: Davidson's principles and practice of medicine (18th ed). Churchill Livingstone, London, 1999; PP. 322-331. 9. Abbas, A.K., Lichtman, A.H., Pober, J.S.: Cellular and Molecular Immunology (2nd ed.), W.B. Saunders, Philadelphia, 1994; PP.198-205. 10. Elgert, K.D.: Immunology: Understanding the Immune System. New York, Wiley-Liss. 1996. 11. Barrett, K.E.: Cytokines: sources, receptors, and signaling. Bailliers Clinc. Gastroenterol. 1996; 10: 1-15. Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Evaluation of the role of IL-2 and IL-4 in asthma 53 12. Roback, T.: Biological properties and therabeutic use of interleukin-2 (IL-2). Postepy. Hig. Med. Dosw. 1995; 49: 367- 393. 13. Sigal, L.H. and Ron,W.: Immunology and inflammation: Basic mechanisms and clinical consequences. New York, Mc Graw-Hill. 1994. 14. Gerald, W.; Volcheck, M.D.; James, T.C.; Li, M.D.: Mechanisms of Asthma: The Role of Neurokinins, Nitric Oxide, and Genetics. Medscape General Medicine 1999; 1(3). 15. Vignola, A.M.; Chanez, P. and Bousquet, J.: Management of severe asthma. In: Severe Asthma: Pathogenesis and Clinical Management. Szefler, S.J. and Leung, D.Y.M. (Eds.). Marcel Dekker, New York, 2001; PP. 575-596. 16. Lee, T.H.; Brattsand, R. and Leung, D.Y.M.: Corticosteroid action and resistance in asthma. Am. J. Respir. Cell Mol. Biol. 1996; 154 (Suppl): S1-S79. 17. Donald, Y.M.L.; Joseph, D.S. and Stanly, J.S.: Steroid-Unresponsive Asthma. Semin. Respir. Crit. Care Med. 2002; 23(4): 387-398. 18. Leung, D.Y.M.; Martin, R.J.; Szefler, S.J.; Sher, E.R.; Ying, S.; Kay, A.B. and Hamid, Q.: Dysregulation of interleukin 4, interleukinي,5يandيinterferonيγيgeneي expression in steroid-resistant asthma. J. Exp. Med. 2003; 181: TK. 19. Corrigan, C.J.; Brown, P.H. and Barnes, N.C.: Glucocorticoid resistance in chronic asthma: glucocorticoid pharmacokinetics, glucocorticoid receptor characteristics, and inhibition of peripheral blood T cell proliferation by glucocorticoids in vitro. Am. Rev. Respir. Dis. 144: 1991; 1016- 1025 20. Alvarez, J.; Surs, W.; Leung, D.Y.M.; Ikle, D.; Gelfand, E.W. and Szefler, S.J.: Steroid-resistant asthma: immunologic and pharmacologic features. J. Allergy Clin. Immunol. 1992; 89: 714-721. 21. Yang-Yen, H.F.; Chambard, J.C. and Sun, Y.L.: Transcriptional interference between c-jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 1990; 62: 1205-1215. 22. Adcock, I.M.; Lane, S.J.; Brown, C.R.; Peters, M.J.; Lee, T.H. and Barnes, P.J.: Differences in binding of glucocorticoid receptor to DNA in steroid-resistance asthma. J. Immunol. 1995; 154: 3500- 3505. 23. Bamberger, C.M.; Bamberger, A.M.; deCastro, M. and Chrousos, G.P.: Glucocorticoid receptor beta, a potential endogenous inhibitor of glucocorticoid action in humans. J. Clin. Invest. 1995; 95: 2435-2441. 24. Oakley, R.H.; Jewell, C.M.; Yudt, M.R.; Bofetiado, D.M. and Cidlowski, J.A.: The dominant negative activity of the human glucocorticoid receptor beta isoform. Specifity and mechanism of action. J. Biol. Chem. 1999; 274: 27857-27866. 25. Vottero, A. and Chrousos, G.P.: Glucocorticoid receptor beta: view I. Trends Endocrinol. Metab. 1999; 10: 333-338. 26. Nimmagadda, S.R.; Szefler, S.J.; Spahn, J.D.; Surs, W. and Leung, D.Y.M.: Allergen exposure decreases glucocorticoid receptor binding affinity and steroid responsiveness in atopic asthmatics. Am. J. Respir. Crit. Care Med. 1997; 155: 87-93. 27. Hauk, P.J.; Wenzel, S.E.; Trumble, A.E.; Szefler, S.J. and Leung, D.Y.M.: Increased T cell receptor vbeta8+ T cells in bronchoalveolar lavage fluid of subjects with poorly controlled asthma: a potential role for microbial superantigens. J. Allergy Clin. Immunol. 1999; 104: 37- 45. 28. Didierjean, L.; Salomon, D. and Merot, Y.: Localization and characterization of the interleukin 1 immunoreactive pool (IL-1 alpha and beta forms) in normal human epidermis. J. Invest. Dermatol. 1989; 92: 809-816. 29. Kyle, R.A.: Classification and diagnosis of monoclonal gammopathies. In: Manual of clinical laboratory immunology (3rd ed.), Rose, N.R.; Friedman, H. and Fahey, J.L. (Eds.), Washington, DC: American Society for Microbiology, 1986; pp. 152. 30. Ishioka, S.; Terada, M.; Haruta, Y.; Hiyama, K.; Hozawa, S. and Yamakido, M.: Multiple logistic regression analysis of risk factors for the development of steroid-dependant asthma in the elderly: a comparisem with younger asthmatics. Respiration 2001; 68(1): 35-40. 31. Chalmers, G.W.; Macleod, K.J.; Little, S.A.; Thomson, L.J.; McSharry, C.P. and Thomson, N.C.: Influence of cigarette smoking on inhaled corticosteroid treatment in mild asthma. Thorax. Mar. 2002; 57(3): 226-30. 32. Horvath, I.; Donnelly, L.E.; Kiss, A.; Balint, B.; Kharitonov, S.A. and Barnes, P.J.: Exhaled nitric oxide and hydrogen peroxide concentrations in asthmatic smokers. Respiration 2004; 71(5): 463-8. Iraqi J.Pharm.Sci., Vol.17 (1) ,2008 Evaluation of the role of IL-2 and IL-4 in asthma 54 33. Busse, W.; Corren, J. and Lanier, B.Q.: Omalizumab, anti-IgE recombinant humanized monoclonal antibody, for the treatment of severe allergic asthma. J. Allergy Clin. Immunol. 2001; 108: 184- 190. 34. Nathan, R.; and Erwin, W.G.: Severe Steroid-Dependent Asthma: Therapeutic Role of High-Dose Intravenous Immunoglobulin. Medscape General Medicine 2000; 2(1). 35. Burchard, E.G.; Silverman, E.K. and Rosenwasser, L.J.: Association between a sequence variant in the IL-4 gene promoter and FEV(1) in asthma. Am. J. Respir. Crit. Care Med. 1999; 160: 919- 922. 36. Leung, D.Y.M.; Hamid, Q. and Vottero, A.: Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta. J. Exp. Med. 1997; 136: 23-28. 37. Hamid, Q.A.; Wenzel, S.E. and Hauk, P.J.: Increased glucocorticoid receptor beta in airway cells of glucocorticoid- insensitive asthma. Am. J. Respir. Crit. Care Med. 1999; 159(5 Pt 1): 1600-1604. 38. Liao, W.J.; Li, Y.M.; Chen, T.; He, W.Q.; Lin, Y.P. and Li, N.: Determination of serum acute phase reaction protein in patients with severe acute respiratory syndrome. Zhonghua Yu Fang Yi Xue Za Zhi 2004; 38(2): 92-3. 39. Kam, J.C.; Szefler, S.J.; Surs, W.; Sher, E.R. and Leung, D.Y.: Combination IL-2 and IL-4 reduces glucocorticoid receptor- binding affinity and T cell response to glucocorticoids. J. Immunol. 1993; 151: 3460-3466. 40. Zubiaga, A.M.; Munoz, E. and Huber, B.T.: IL-4 and IL-2 selectively rescue subsets from glucocorticoid-induced apoptosis. J. Immunol. 1992; 149: 107- 112. 41. Goleva, E.; Kisich, K.O. and Leung, D.Y.M.: A role for STAT5 in the pathogenesis of IL-2-induced glucocorticoid resistance. J. Immunol. 2002; 169: 5934-5940. 42. Rosa-Rosa, L.; Zimmermann, N.; Bernstein, J.A.; Rothenberg, M.E. and Khurana, H.G.K.: The R576 IL-4 receptor alpha allele correlates with asthma severity. J. Allergy Clin. Immunol. 1999; 104: 1008-1014. 43. Spahn, J.D.; Landwehr, L.P.; Nimmagadda, S.; Surs, W.; Leung, D.Y.M. and Szefler, S.J. (1996): Effects of glucocorticoids on lymphocyte activation in patients with steroid- sensitive and steroid-resistant asthma. J. Allergy. Clin. Immunol. 98(6 Pt 1): 1073- 1079.