IgE sensitization to food allergens and airborne allergens in relation to biomarkers of type 2 inflammation in asthma.

BACKGROUND: We have recently reported that sensitization to food allergens and sensitization to airborne allergens had independent associations with increased fraction of exhaled nitric oxide (FeNO) and blood eosinophils in middle-aged adults and in young subjects with asthma. OBJECTIVE: To investigate the relation between IgE sensitization and several type 2 inflammation biomarkers in adult asthmatics. METHODS: FeNO, urinary eosinophil-derived neurotoxin (U-EDN), serum eosinophil cationic protein (S-ECP) and periostin were measured in 396 asthmatics, aged 17-76 years, from the Swedish GA2LEN study. Sensitization to airborne allergens was examined with skin prick tests (≥3 mm wheal) and sensitization to food allergens with measurement of specific IgE (≥0.35 kU/L). RESULTS: Asthmatics sensitized to food allergens had higher FeNO, 22.3 ppb (18.6, 26.7) vs 16.1 ppb (14.2, 18.2) (P = .005), S-ECP, 17.7 mg/L (14.8, 21.1) vs 12.8 mg/L (10.9, 14.9) (P = .01), and periostin, 73.7 (67.5, 80.3) ng/mL vs 59.9 (55.8, 64.2) ng/mL (P = .003), than non-sensitized subjects. Periostin levels in this group were also significantly higher than in the group sensitized only to airborne allergens (P = .01). Sensitization to food allergens related independently to FeNO (P = .02), S-ECP (P = .006) and periostin (P = .004), whereas sensitization only to airborne allergens related only to FeNO (P = .02) after adjustments for age, sex, height, weight and smoking history. FeNO correlated weakly with S-ECP (r = .17, P < .001), periostin (r = .19, P < .001) and U-EDN (0.16, P < .001). S-ECP also correlated weakly with U-EDN (r = .12, P = .02). None of the correlations between the remaining pairs of markers of type 2 inflammation were significant. CONCLUSIONS & CLINICAL RELEVANCE: Sensitization to food allergens related to several local and systemic type 2 inflammation markers, such as FeNO, S-ECP and periostin. Assessing the profile of allergic sensitization, including to food allergens, might improve the understanding and interpretation of inflammatory markers and potentially improve asthma management.

Serum periostin relates to type-2 inflammation and lung function in asthma: Data from the large population-based cohort Swedish GA(2)LEN.

BACKGROUND: Periostin has been suggested as a novel, phenotype-specific biomarker for asthma driven by type 2 inflammation. However, large studies examining relationships between circulating periostin and patient characteristics are lacking and the suitability of periostin as a biomarker in asthma remains unclear. AIM: To examine circulating periostin in healthy controls and subjects with asthma from the general population with different severity and treatment profiles, both with and without chronic rhinosinusitis (CRS), in relation to other biomarkers and clinical characteristics. METHODS: Serum periostin was examined by ELISA in 1100 subjects aged 17-76 from the Swedish Global Allergy and Asthma European Network (GA(2)LEN) study, which included 463 asthmatics with/without chronic rhinosinusitis (CRS), 98 individuals with CRS only, and 206 healthy controls. Clinical tests included measurement of lung function, Fraction of exhaled NO (FeNO), IgE, urinary eosinophil-derived neurotoxin (U-EDN), and serum eosinophil cationic protein (S-ECP), as well as completion of questionnaires regarding respiratory symptoms, medication, and quality of life. RESULTS: Although median periostin values showed no differences when comparing disease groups with healthy controls, multiple regression analyses revealed that periostin was positively associated with higher FeNO, U-EDN, and total IgE. In patients with asthma, an inverse relationship with lung function was also observed. Current smoking was associated with decreased periostin levels, whereas increased age and lower body mass index (BMI) related to higher periostin levels in subjects both with and without asthma. CONCLUSION: We confirm associations between periostin and markers of type 2 inflammation, as well as lung function, and identify novel constitutional factors of importance to the use of periostin as a phenotype-specific biomarker in asthma.

Elevation of serum squamous cell carcinoma antigen 2 in patients with psoriasis: associations with disease severity and response to the treatment.

BACKGROUND: Squamous cell carcinoma antigen (SCCA) belongs to the ovalbumin-serpin family and is a known tumour marker. Expression of SCCA is upregulated in the serum and skin of patients with psoriasis. OBJECTIVES: The aim of this study was to determine SCCA2 levels in association with disease severity and treatment efficacy in patients with psoriasis. MATERIALS AND METHODS: Patients with psoriasis (n = 123) and healthy controls (n = 25) were enrolled in this prospective cross-sectional study. Enzyme-linked immunosorbent assay (ELISA) analysis was performed to determine serum SCCA2 levels. SCCA2 expression in skin was evaluated using immunohistochemical analysis. Serum SCCA2 levels were compared with Psoriasis Area Severity Index (PASI) scores. The effect of treatment on serum SCCA2 levels was assessed using serial examinations. Induction of SCCA2 by several psoriatic cytokines in human keratinocytes was evaluated. RESULTS: The serum levels of SCCA2 were significantly higher in patients with psoriasis than healthy controls and correlated well with disease severity. Increased SCCA2 staining was observed in lesional skin but not in nonlesional skin of patients with psoriasis. In addition, SCCA2 expression levels in skin correlated with serum concentrations of SCCA2. SCCA2 significantly decreased according to improvement of PASI scores. Interleukin (IL)-17 and IL-22 synergistically increased the production of SCCA2 at both mRNA and protein levels in human keratinocytes. CONCLUSIONS: Significant elevation of SCCA2 is associated with disease severity and reflects treatment efficacy. SCCA2 may be a useful biomarker in psoriasis, reflecting T-helper 17-type inflammation - the main determinant of the severity of psoriasis.

GLCCI1 variant accelerates pulmonary function decline in patients with asthma receiving inhaled corticosteroids.

BACKGROUND: In steroid-naive patients with asthma, several gene variants are associated with a short-term response to inhaled corticosteroid (ICS) treatment; this has mostly been observed in Caucasians. However, not many studies have been conducted for other ethnicities. Here, we aimed to determine the relationship between the annual decline in forced expiratory flow volume in one second (FEV1 ) and the variant of the glucocorticoid-induced transcript 1 gene (GLCCI1) in Japanese patients with asthma receiving long-term ICS treatment, taking into account the effect of high serum periostin levels, a known association factor of pulmonary function decline and a marker of refractory eosinophilic/Th2 inflammation. METHODS: In this study, 224 patients with asthma receiving ICS treatment for at least 4 years were enrolled. The effects of single-nucleotide polymorphisms (SNPs) in GLCCI1, stress-induced phosphoprotein 1 (STIP1), and T gene on the decline in FEV1 of 30 ml/year or greater were determined. RESULTS: Besides the known contributing factors, that is, the most intensive treatment step, ex-smoking, and high serum periostin levels (≥95 ng/ml), the GG genotype of GLCCI1 rs37973, and not other SNPs, was independently associated with a decline in FEV1 of 30 ml/year or greater. When patients were stratified according to their serum periostin levels, the GG genotype of rs37973 was significantly associated with blood eosinophilia (≥250/µl) in the high serum periostin group. CONCLUSIONS: A GLCCI1 variant is a risk factor of pulmonary function decline in Japanese patients with asthma receiving long-term ICS treatment. Thus, GLCCI1 may be associated with response to ICS across ethnicities.

A notable case report of May-Hegglin anomaly with immune complex-related nephropathy: a genetic and histological analysis.

May-Hegglin anomaly (MHA) is a rare autosomal dominant disease characterized by macrothrombocytopenia and leukocyte inclusions with microfilaments in the ribosomes. Mutations in the MYH9 gene, encoding non-muscle myosin heavy chain IIA (NMMHC-IIA) have been identified in patients with MHA and other MYH9-related diseases. Two young males (an older and younger brother) presented with macrothrombocytopenia and leukocyte inclusion bodies. Electron microscopy (EM) revealed parallel filaments in leukocyte inclusion bodies characteristic of MHA. Immunofluorescence microscopy (IF) showed NMMHC-IIA antibodies in 1 - 2 leukocyte inclusion bodies. These findings were consistent with MHA and they were identified to express the MYH9 mutation, D1424H. The older brother underwent a renal biopsy because of persistent proteinuria. Histology revealed mesangial proliferative glomerulonephritis with granular deposits of IgG and C1q. EM showed that the dense deposits were located in subendothelial cells, mesangial cells and Bowman's capsule. Immunocytochemistry revealed that NMMHC-IIA antibodies were localized in podocyte and endothelial cells in the glomerulus. Moreover, the expression of nephrin and podocin, slit diagram protein, was normal. An inflammatory mechanism may occur separately from MYH9-related disease. This report presents a case of MHA with immune complex-related nephropathy.

Recent progress in understanding the diversity of the human ov-serpin/clade B serpin family.

The inhibitory mechanism against proteases is important in the maintenance of homeostasis or health in the body. The human ovalbumin serpin (ov-serpin)/clade B serpin family is one group of the human serpins, a family of serine protease inhibitors. They have acquired diversity in the profiles of target proteases, inhibitory mechanisms, and localization patterns during their evolution. Most serpins target serine proteases, however, some ov-serpins target only cysteine proteases or both serine and cysteine proteases and furthermore, several ov-serpins do not possess inhibitory activities. Although the ov-serpins act primarily as intracellular serpins, some show extracellular and nuclear localizations. Such diversity enables the ov-serpins to play multiple physiological roles in the body. Recent analyses have revealed that the functions of human ov-serpins are more diversified than we previously knew. In this article, we describe recent progress in our understanding of how the human ov-serpin/clade B serpin family demonstrates diversity.

The squamous cell carcinoma antigens as relevant biomarkers of atopic dermatitis.

BACKGROUND: Although it is thought that both Th1- and Th2-type inflammations are involved in the pathogenesis of atopic dermatitis (AD), it is controversial which immune response is more involved in regulating the clinical severity of AD. We recently found that the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2 are novel biomarkers of bronchial asthma, downstream of IL-4 and IL-13. OBJECTIVE: We examined whether SCCA1 and SCCA2 could also serve as biomarkers of AD, reflecting its Th2-type immune responses, and whether the expression level of SCCA was correlated with clinical severity of AD. METHOD: We compared the expression of SCCA1 and SCCA2 at the mRNA and protein levels in both involved and uninvolved skin of AD patients and in normal control skin. We next analysed induction of SCCA by IL-4 or IL-13 in keratinocytes. Finally, we compared the serum level of SCCA with laboratory parameters reflecting Th2-type inflammation and clinical severity in AD patients. RESULTS: SCCA1 and SCCA2 were highly expressed in involved skin of AD patients, compared with their uninvolved skin, at both mRNA and protein levels. SCCA protein was dominantly expressed in suprabasal keratinocytes in the epidermis of AD patients. Either IL-4 or IL-13, but not IFN-gamma or TNF, induced production of SCCA in keratinocytes. These result suggest that SCCA is induced in AD skin, probably due to direct actions of IL-4 and/or IL-13 on keratinocytes. Serum levels of SCCA were well correlated with eosinophil numbers and serum lactate dehydrogenase levels, and weakly with serum IgE levels, in AD patients. Furthermore, serum levels of SCCA were strongly correlated with clinical severity. CONCLUSIONS: Th2-type inflammation dominantly regulates the clinical severity of AD, and SCCA is a relevant biomarker of AD, reflecting both Th2-type inflammation and clinical severity.

Characterization of novel squamous cell carcinoma antigen-related molecules in mice.

The squamous cell carcinoma antigen 1 (SCCA1) and SCCA2 are unique serpins that can inhibit cysteine proteinases. SQN-5, their mouse ortholog, has already been identified, and its inhibitory property has been characterized; however, its biological role has remained undefined. Furthermore, no other mouse homolog of SQN-5 has been known. We characterize three mouse members of SCCA-related molecules including SQN-5 in this article. Serpinb3a (SQN-5) and Serpinb3b, but not Serpinb3c, were functional, inhibiting both serine and cysteine proteinases with different inhibitory profiles due to the difference of two amino acids in their reactive site loops. Serpinb3a was ubiquitously expressed in most tissues, whereas expression of Serpinb3b was limited to keratinocytes. Keratinocytes secreted both SCCA-related proteins, Serpinb3a and Serpinb3b. These results indicate that Serpinb3a and Serpinb3b may play different roles by inhibiting intrinsic or extrinsic proteinases with different expression distributions and different inhibitory profiles.

PKCdelta and zeta mediate IL-4/IL-13-induced germline epsilon transcription in human B cells: a putative regulation via PU.1 phosphorylation.

We have investigated the role of PKC isozymes in the function of IL-4 and IL-13 in human B cells. In a Burkitt's B lymphoma cell line, DND39, IL-4 induced the translocation of PKCdelta and zeta from the cytosol to the membrane fraction. The activation of germline epsilon promoter by IL-4 was abrogated not only by the expression of dominant negative mutants of PKCdelta and zeta but also by isozyme-selective PKC inhibitors, rottlerin and PKCzeta pseudosubstrate peptide. These inhibitors also suppressed IL-4/IL-13-induced germline epsilon transcription in the IL-13Ralpha1-transfected DND39 cells as well as in normal human B cells, but had no influence on the induction of CD23b in the latter cells. As a downstream event of PKC, we found threonine phosphorylation of PU.1 in IL-4-stimulated DND39 cells. This phosphorylation was suppressed by the PKC inhibitors, although STAT6 activation was unaffected. These results suggest that, in human B cells, IL-4/IL-13 utilize PKCdelta and zeta for the STAT6-independent signaling pathway and thereby modulate the transcriptional activity of PU.1.

Interleukin-13 and interleukin-13 receptor in Hodgkin's disease: possible autocrine mechanism and involvement in fibrosis.

AIMS: Hodgkin's disease (HD) is characterized by the presence of Hodgkin and Reed-Sternberg (H-RS) cells against a hyperplastic background of reactive cells such as lymphocytes, histiocytes, plasma cells, eosinophils, neutrophils, and stromal cells. There is ample evidence to suggest that proliferation and survival of HD-derived cells is due to cytokine signalling. Recently, high expression of interleukin (IL)-13 was described in HD-derived cell lines. Here we investigated the possible involvement of IL-13 in the pathophysiology, especially autocrine pathways of H-RS cells. METHODS AND RESULTS: The expression of IL-13 and IL-13 receptor (IL-13R) was determined by immunostaining and reverse transcriptase-polymerase chain reaction in 39 cases of HD, including 17 cases with nodular sclerosis (NS) type, 19 cases with mixed cellularity (MC), and three cases with lymphocyte predominance (LP) type. Expression of IL-13 was confined to H-RS cells and a few lymphocytes. IL-13R was expressed in H-RS cells, lymphocytes, histiocytes, fibroblasts, and endothelial cells. H-RS cells of MC and NS types frequently expressed both IL-13 and IL-13R. However, the number of IL-13-positive H-RS cells was statistically higher in NS-type than in MC-type, but the number of IL-13R was similar. IL-13R-positive fibroblasts were frequently encountered in NS-type. H-RS cells of LP type rarely expressed IL-13. CONCLUSIONS: Our results suggest that IL-13 might be involved in autocrine pathways of H-RS cells and fibrosis at least in NS-type. Our results also indicated that in addition to the morphological and phenotypic differences, the neoplastic cells of LP type might be functionally different from H-RS cells of MC- and NS-types.

Characterization of IL-4 and IL-13 signals dependent on the human IL-13 receptor alpha chain 1: redundancy of requirement of tyrosine residue for STAT3 activation.

IL-4 and IL-13 are pleiotropic cytokines whose biological activities overlap with each other. IL-13 receptor alpha chain 1 (IL-13R alpha 1) is necessary for binding to IL-13, and the heterodimer composed of IL-13R alpha 1 and IL-4R alpha chain transduces IL-13 and IL-4 signals; however, the functional mapping of the intracellular domain of IL-13R alpha 1 is not fully understood. In this study, we constructed wild and mutated types of human IL-13R alpha 1, and analyzed IL-4 and IL-13 signals using an IL-13R alpha 1-transfected human B cell line. Expression of IL-13R alpha 1 evoked STAT3 activation by IL-4 and IL-13, and in stimulated human B cells, on which IL-13R alpha 1 was highly expressed, IL-4 and IL-13 induced STAT3 activation. Replacement of the two tyrosine residues completely abolished STAT3 activation, although replacing either tyrosine residue alone retained it. Furthermore, we found that the Box1 region and the C-terminal tail of IL-13R alpha 1 were critical for binding to Tyk2, and activation of Jak1, Tyk2, the insulin receptor substrate-1 and STAT6 respectively. These results suggest that STAT3 activation is involved with IL-4 and IL-13 signals in human B cells along with the activation of STAT6, and that there is a unique sequence in IL-13R alpha 1 to activate STAT3.

Augmentation of signal transducer and activation of transcription (STAT)6 and STAT3 expression in stimulated B and T cells.

Signal transducer and activation of transcription (STAT)6 has a central role in the signal transduction of interleukin (IL)-4 and IL-13. It has recently been revealed that STAT3 is also involved. STAT6 and STAT3 are expressed ubiquitously; however, it remains unknown how STAT6 and STAT3 expression is regulated. In this study, we found that STAT6 expression was augmented at the transcription level in B and T cells stimulated with anti-IgM antibody and anti-CD40 antibody or PMA and ionomycin, respectively, and that STAT3 expression was similarly augmented in the stimulated B cells. The stimulated B and T cells showed enhancement of STAT6 activation and CD23 expression induced by IL-4 and IL-13. Augmentation of STAT6 and STAT3 would be a mechanism of the amplification of the IL-4 and IL-13 signals in stimulated B and T cells.

Lipopolysaccharide increases cyclo-oxygenase-2 expression in a colon carcinoma cell line through nuclear factor-kappa B activation.

Both nonneoplastic colon epithelium and colon carcinoma cells in situ are continuously exposed to lipopolysaccharide (LPS). Few reports have addressed possible direct effects of LPS in promotion of colon carcinoma and underlying mechanisms. We found evidence that LPS directly stimulated growth of the human colon carcinoma cell line CE-1 through an increase in the production of prostaglandin E2 (PGE2) as a result of cyclo-oxygenase-2 (COX-2) expression. LPS induced significant increases in PGE2 production in CE-1 cells, which were found to express a high-affinity LPS receptor, CD14. Positive correlations were found between PGE2 production and activation of nuclear factor (NF)-kappa B as well as expression of both COX-2 mRNA and protein in LPS-stimulated CE-1 cells. When CE-1 cells were exposed to exogenous PGE2, DNA synthesis increased. These results indicate that LPS may stimulate DNA synthesis in certain colon carcinoma cells as a result of PGE2 production involving increased COX-2 expression that might result in turn from activation of NF-kappa B by LPS. Further investigation of the pathways mediating LPS-induced stimulation of colon carcinoma cells may provide insights into LPS effects in in vivo tumor biology.

Antagonistic effects of an alternative splice variant of human IL-4, IL-4delta2, on IL-4 activities in human monocytes and B cells.

IL-4 is a pleiotropic cytokine which exerts its actions on various lineages of hematopoietic and nonhematopoietic cells. This cytokine is one of the central regulators of immunity in health and disease states. An alternative splice variant, in which the second of four exons is omitted, has been recently described and designated as IL-4delta2. The variant has been previously described as a potential naturally occurring antagonist of human IL-4 (hIL-4)-stimulated T cell proliferation. In this study, we investigated the effects of recombinant human (rh) IL-4delta2 on monocytes and B cells. In monocytes, rhIL-4delta2 blocked inhibitory action of hIL-4 on LPS-induced cyclooxygenase-2 expression and subsequent prostaglandin E2 secretion. In B cells, rhIL-4delta2 was an antagonist of the hIL-4-induced synthesis of IgE and expression of CD23. Our results broaden the spectrum of hIL-4-antagonistic activities of rhIL-4delta2, thus creating the background for the potential use of rhIL-4delta2 as a therapeutic anti-hIL-4 agent.

Differential effects of interleukin-4 and interleukin-10 on nitric oxide production by murine macrophages.

OBJECTIVE: To study the effect of interleukin (IL)-4 and IL-10 on nitric oxide (NO) production by macrophages. MATERIALS AND METHODS: Elicited or resident peritoneal macrophages (PMO) and a macrophage cell line Raw264.7 were primed by IL-4 or IL-10 for 6 hours, and were further incubated in the presence of interferon (IFN)-gamma and/or lipopolysaccharide (LPS) for 48 hours. NO2- accumulation in the supernatant of cultured cells was used as an indicator of NO production and was determined by the standard Griess reaction adapted for microplates. The amount of tumor necrosis factor (TNF)-alpha in the culture supernatants was determined with a commercially available ELISA kit. The absorbance was measured at 450 nm with a microplate photometer. RESULTS: IL-4 inhibited NO production by murine macrophages of different sources and the macrophage cell line Raw264.7. In contrast, different macrophage populations showed differential responses to IL-10. After stimulation with LPS or IFN-gamma, IL-10 suppressed NO production by elicited PMO but enhanced NO production by resident PMO or by Raw264.7. Both IL-4 and IL-10 inhibited the production of TNF-alpha, which has been shown to play a crucial role in NO production. In the presence or the absence of blocking antibody to TNF-alpha, IL-10 always enhanced NO production by resident PMO. This result suggests that the inhibition of TNF-alpha production and the enhancement of NO production by resident PMO stimulated with IL-10 are independent, coexisting events. CONCLUSIONS: Factors other than TNF-alpha have been suspected to influence NO production by macrophages, and this study indicates that IL-10 may be a candidate cytokine for resident PMO.

Activation of STAT5 by lipopolysaccharide through granulocyte-macrophage colony-stimulating factor production in human monocytes.

LPS is a potent stimulator of monocytes, inducing many of their functions. Although the details of how LPS exerts such functions remain largely unknown, transcription factors such as nuclear factor-kappaB, nuclear factor-IL-6, and activator protein-1 have been shown to be involved in this process. However, to date it has been thought that no known STAT molecule plays a role in the activation of monocytes by LPS. In this study we examined whether some known STAT molecule is stimulated by LPS, based on the finding that a GAS motif sequence is conserved in the promoter regions of human, mouse, and rat cyclo-oxygenase-2 (COX-2) genes. Consequently, LPS induced activation of STAT5 in human monocytes, and this STAT5 activation occurred in an indirect way via granulocyte-macrophage CSF (GM-CSF) secreted by LPS-stimulated monocytes. Expression of COX-2 protein was partially reduced by treatment of anti-human GM-CSF Ab. Activation of STAT5 was inhibited by either IL-10 or dexamethasone (Dex), but not by aspirin. IL-10 blocked activation of STAT5 indirectly by suppressing GM-CSF production, while Dex inhibited this activation both directly and indirectly. Taken together, these results suggest that in addition to other transcription factors, STAT5 plays an important role in activation of monocytes by LPS, and that STAT5 is another target for IL-10 and Dex to inhibit COX-2 expression in activated monocytes.

Constitutive activation of Jak-2 and Tyk-2 in a v-Src-transformed human gallbladder adenocarcinoma cell line.

It is well known that v-Src phosphorylates various substrates on tyrosine residue and associates with tyrosine-phosphorylated proteins as well as proline-rich ligands through its SH2 and SH3 domains, respectively, thereby inducing oncogenic transformation. A signal pathway from the cell surface to genes in the nucleus, the Jak/STAT (signal transducers and activators of transcription) pathway, has been shown to be involved in the signal transduction mechanism mediated by many cytokines and growth factors. Although a member of the STAT family, STAT3 has been reported to be constitutively activated in several v-Src-transformed cells, and it still remains unknown whether Jak molecules, which act upstream of STATs, are involved in the v-Src-induced activation mechanism of STAT3. In this study, we analyzed activations of both Jak and STAT molecules using v-Src-transformed HAG-1 cells derived from a human gallbladder adenocarcinoma. STAT3 was found to be constitutively activated in v-Src-transformed HAG-1 cells, but not in either non-transformed mock-transfected or activated c-H-ras-transfected HAG-1 cells, even though the other known STAT molecules are expressed. Furthermore, both Jak-2 and Tyk-2 were constitutively activated only in v-Src-transformed HAG-1 cells. Association of v-Src with either STAT3 or the Jak molecules was not observed. No change of this activation was detected by either interferon (IFN)-alpha2a or IFN-gamma, which had shown inhibitory effects on the growth of v-Src-transformed HAG-1 cells. These results raise the possibility that Jak-2 and Tyk-2 are both activated by v-Src, thereby contributing to the constitutive activation of STAT3 in the v-Src-transformed cells.

Interleukin-4 activates two distinct pathways of phosphatidylinositol-3 kinase in the same cells.

We have recently demonstrated that c-fes protooncogene product (FES), or a FES-related protein, associates with the interleukin-4 receptor alpha chain (IL-4R alpha) and phosphatidylinositol-3 (PI3) kinase in mouse T cell lines; however, others have demonstrated that PI3 kinase associates with IL-4R alpha through tyrosine phosphorylated insulin receptor substrate (IRS)-2 in other cell types. In order to examine whether IL-4 activates these two distinct PI3 kinase pathways in the same cells, we analyzed association of PI3 kinase with IRS-2, and tyrosine phosphorylation of IRS-2, in a mouse pro-B cell line, Ba/F3, and a mouse mast cell line, MC9. In both cell lines, IL-4 induced tyrosine phosphorylation of IRS-2, association of PI3 kinase with IRS-2, and FES or a FES-related protein. These results indicate that IL-4 activates two distinct PI3 kinase pathways in the same cells. We further identified the critical region in the cytoplasmic domain of IL-4R alpha required for tyrosine phosphorylation of IRS-2.

Interleukin-4 induces association of the c-fes proto-oncogene product with phosphatidylinositol-3 kinase.

We have previously demonstrated that interleukin-4 (IL-4) induces tyrosine phosphorylation of a protein closely related or identical to the c-fes proto-oncogene product (FES) and association of this protein with the IL-4 receptor alpha chain (IL-4R alpha). IL-4 is known to induce association of phosphatidylinositol-3 (PI3) kinase with the IL-4R alpha. Since FES contains the consensus motifs for PI3 kinase binding, we tested the possibility that FES may associate with PI3 kinase upon IL-4 stimulation. We demonstrate herein that IL-4 stimulation induced rapid association of FES or a related protein with PI3 kinase in mouse T-cell lines. We also show an association of human FES (hFES) with the src homology 2 (SH2) domain of PI3 kinase in a COS7 cell expression system. The in vitro PI3 kinase assay using COS7 cells suggested that hFES partly contributes to the association between the hIL-4R alpha and PI3 kinase. We have further identified the important region in the cytoplasmic domain of the hIL-4R alpha for association of tyrosine-phosphorylated hFES with the hIL-4R alpha and SH2 domain of PI3 kinase using a COS7 cell expression system. These results suggest that FES or a related protein/PI3 kinase pathway may play a role in the pleiotropic effects of IL-4.

The critical region in the cytoplasmic domain of human IL-4 receptor for induction of IgE synthesis.

To examine the region critical for differentiation in the human IL-4 receptor (hIL-4R), we transfected the Abelson murine leukemia virus (A-MuLV)-transformed murine pre-B cell line A20 with plasmid DNA encoding the hIL-4R. Transfectants expressed high affinity hIL-4Rs on the cell surface. Treatment with LPS and hIL-4 induced germline C epsilon transcripts in hIL-4R expressing A20 cells. Several hIL-4R mutant plasmids were then transfected into A20 cells and the transfectants were examined for hIL-4R expression and the ability to induce germline C epsilon transcripts upon stimulation with LPS and hIL-4. Although all A20 transfectants tested expressed the high-affinity hIL-4R, A20 transfectants expressing the mutant hIL-4R, which contains only 8 amino acids in the cytoplasmic domain, did not respond to LPS and hIL-4 with germline C epsilon transcripts. In addition, A20 transfectants expressing an internally deleted hIL-4R, in which the deleted region has been identified as the critical region for growth signal transduction in the previous study, failed to induce germline C epsilon transcripts with LPS and hIL-4. These results indicate that the critical region for the differentiation signal in the hIL-4R is identical to that for the growth signal, suggesting that IL-4 may share, at least partly, a common signal pathway for both growth and differentiation.