The role of interleukin 4 and IL-4RA in intervertebral disc degeneration: investigation of single nucleotide polymorphisms in genes and a systematic review & meta-analysis of IL-4 expression level.

Background: Intervertebral disc degeneration (IVDD) is a multifactorial disease that is sensitive to the balance between anti-inflammatory and pro-inflammatory cytokines. This study investigated the single nucleotide polymorphisms (SNPs) of interleukin 4 (IL-4) in IVDD.Methods: Genomic DNA of peripheral mononuclear cells of 76 IVDD patients and 140 healthy controls were investigated for three SNPs of IL-4 (rs2243248 (-1098G/T), rs2243250 (-590 C/T), rs2070874 (-33 C/T)) and 1 SNP of IL-4RA (rs180275, +1902 A/G) through PCR-SSP method.Results: The 'C' allele frequency of IL-4 rs2243250 was 104 in 76 patients, while it was 149 in 140 controls (OR = 2, p = .001); also this SNP was significantly associated with post-operative pain reduction. The 'C' allele of IL-4 rs2070874 (130 in 76 patients, and 200 in 140 controls, OR = 2.66), and the 'CC' genotype were more frequent among patients (OR = 3.98, p < .001) than controls. 'TTT' haplotype was more common in controls (OR = 0.36, p < .001) and 'TCC' was also more common in patients (OR = 1.75, p = .012). A meta-analysis of previous studies found significantly higher IL-4 levels in disc tissues of IVDD patients, which was not similarly found in blood samples.Conclusion: The immune system plays an important role in IVDD. The extent and progress of the disease vary significantly with IL-4 level. Meanwhile, the rs2070874 and rs2243250 SNPs of IL-4 were significantly associated with IVDD in Iranian patients.

Recurrence and cancer-specific survival according to the expression of IL-4Rα and IL-13Rα1 in patients with oral cavity cancer.

BACKGROUND: Interleukin-4 (IL-4) and interleukin-13 (IL-13) are anti-inflammatory and immunomodulatory cytokines that play crucial roles in cancer progression. However, the clinical significance of the expression of these cytokines and their receptors (IL-4R) in oral cavity squamous cell carcinoma (OSCC) is unknown. Therefore, we evaluated the expression of IL-4R in OSCC specimens by immunohistochemistry (IHC) and analysed its relationship to recurrence and survival. METHODS: A total of 186 patients with OSCC were enrolled, and the expression of IL-4Rα and IL-13Rα1 on their primary tumour specimens was evaluated by IHC and correlated to clinicopathologic parameters, recurrence and survival. FINDINGS: High expression of IL-4Rα and IL-13Rα1 was observed in 60 (32.3%) and 165 (88.7%) patients, respectively. IL-4Rα expression was inversely correlated with parameters reflecting primary tumour burden, including tumour size, tumour stage and depth of invasion at the initial diagnosis (P < 0.05). High expression of IL-4Rα also correlated with a greater risk of recurrence (P = 0.002), but was unrelated to cancer-specific survival (CSS, P = 0.118). Conversely, high IL-13Rα1 expression correlated with reduced recurrence (P < 0.001) and increased CSS (P < 0.001) in OSCC patients. INTERPRETATIONS: High expression of IL-4Rα correlated with increased recurrence, while high IL-13Rα1 expression had an inverse relationship to recurrence and CSS in OSCC patients.

Interleukin-4 receptor alpha overexpression in human bladder cancer correlates with the pathological grade and stage of the disease.

Previously, we have demonstrated that interleukin-4 receptor α (IL-4Rα) is overexpressed on a variety of human cancers and can serve as target for IL-4 immunotoxin comprised of IL-4 and a mutated Pseudomonas exotoxin. However, its expression and association with grade and clinical stage of bladder cancer has not been studied. IL-4Rα expression was examined in human bladder cancer cell lines, mouse xenografts, and biopsy specimens at mRNA and protein levels by real-time RT-PCR and IHC/ISH techniques. We also examined the effect of IL-4 on proliferation and invasion of bladder carcinoma cell lines. For tissue microarray (TMA) results, we analyzed the precision data using exact binomial proportion with exact two-sided P-values. We used Cochran-Armitage Statistics with exact two-sided P-values to examine the trend analysis of IL-4Rα over grade or stage of the bladder cancer specimens. The influence of age and gender covariates was also analyzed using multiple logistic regression models. IL-4Rα is overexpressed in five bladder cancer cell lines, while normal bladder and human umbilical vein cell lines (HUVEC) expressed at low levels. Two other chains of IL-4 receptor complex, IL-2RγC and IL-13Rα1, were absent or weakly expressed. IL-4 modestly inhibited the cell proliferation, but enhanced cell invasion of bladder cancer cell lines in a concentration-dependent manner. Bladder cancer xenografts in immunodeficient mice also maintained IL-4Rα overexpression in vivo. Analysis of tumor biopsy specimens in TMAs revealed significantly higher IL-4Rα immunostaining (≥ 2+) in Grade 2 (85%) and Grade 3 (97%) compared to Grade 1 tumors (0%) (P ≤ 0.0001). Similarly, 9% stage I tumors were positive for IL-4Rα (≥ 2+) compared to 84% stage II (P ≤ 0.0001) and 100% stages III-IV tumors (P ≤ 0.0001). IL-13Rα1 was also expressed in tumor tissues but at low levels and it did not show any correlation with the grade and stage of disease. However, the IL-2RγC was not expressed. Ten normal bladder specimens demonstrated ≤ 1+ staining for IL-4Rα and IL-13Rα1 and no staining for IL-2RγC. These results demonstrate that IL-4Rα is overexpressed in human bladder cancer, which correlates with advanced grade and stage of the disease. Thus, IL-4Rα may be a bladder tumor-associated protein and a prognostic biomarker.

Higher TGF-ß with lower CD124 and TSLP, but no difference in PAR-2 expression in bronchial biopsy of bronchial asthma patients in comparison with COPD patients.

Chronic obstructive pulmonary disease (COPD) and bronchial asthma (BA) are 2 severe respiratory disorders with different predominated immunopathologies. There are several "novel molecules" from different families that are proposed as part of the etiopathogenesis of COPD and BA. Proteinase-activated receptor 2 (PAR-2), thymic stromal lymphoprotein (TSLP), interleukin-4 and its receptor (CD124), Yin-Yang 1 (YY1), and transforming growth factor beta (TGF-ß) have been previously shown to be involved in the pathophysiology of both these diseases. We investigated PAR-2, TSLP, CD124 (interleukin-4R), TGF-ß, and YY1 immunohistochemical expression in endobronchial and transbronchial biopsies from 22 BA patients and 20 COPD patients. Immunostaining for the above-mentioned antigens was quantified using a modified semiquantitative scoring system and statistically evaluated. The values of TGF-ß in the epithelial cells (P=0.0007) and TGF-ß in the submucosa (P=0.0075) were higher in the BA samples, whereas values of CD124 (P=0.0015) and TSLP (P=0.0106) were higher in the COPD samples. No statistically significant differences between the groups were recorded for PAR-2 and YY1. Airway inflammatory reaction diversity in BA and COPD seems to be disease specific; however, there are also shared mechanisms involved in the pathophysiology of both diseases.

IL-13 immunotoxin accelerates resolution of lung pathological changes triggered by silica particles in mice.

Instillation of silica into the lungs of rodents results in pathological changes that strongly mimic human silicosis, an occupational lung disease marked by restrictive airway obstruction, inflammation, and fibrosis. Because IL-13 is a pivotal proinflammatory and fibrogenic cytokine, we examined whether a recombinant immunotoxin comprised of human IL-13 and a mutated form of Pseudomonas exotoxin (IL-13-PE) might affect pathological features of experimental silicosis. Mice received a single intranasal instillation of silica particles and were treated with intranasal IL-13-PE every other day from days 21 to 27 postsilica. The sensitivity of putative cell targets to IL-13-PE was also assessed in in vitro settings. Upregulation of IL-13, its receptor subunits IL-13Rα1 and IL-13Rα2, and shared receptor IL-4Rα were associated with development of granulomatous lung inflammation triggered by silica. IL-13-PE inhibited silica-induced granuloma and fibrotic responses noted at 24 h and 15 d after the last treatment. Upregulation of TNF-α, TGF-ß, and chemokines, as well as increased collagen deposition and airway hyperreactivity to methacholine were all clearly sensitive to IL-13-PE. In addition, IL-13-PE inhibited both IL-13-induced proliferation of cultured lung fibroblasts from silicotic mice and silica-induced IL-8 generation from A549 cells. In conclusion, our findings show that therapeutic treatment with IL-13-PE can reverse important pathological features caused by inhalation of silica particles, suggesting that this recombinant immunotoxin is a promising molecular template in drug discovery for the treatment of silicosis.

Lipopolysaccharide-induced interleukin (IL)-4 receptor-α expression and corresponding sensitivity to the M2 promoting effects of IL-4 are impaired in microglia of aged mice.

In several models of aging, microglia become more inflammatory and reactive to immune challenges. For example, peripheral LPS injection causes exaggerated microglial activation associated with prolonged sickness and depressive-like behavior in aged BALB/c mice. Therefore, the purpose of this study was to determine the extent to which age-related amplified microglial activation was associated with reduced sensitivity to the anti-inflammatory and M2 promoting cytokines interleukin (IL)-10 and IL-4. In initial studies with adult mice, LPS induced a time-dependent increase in M1 and M2 mRNA profiles in microglia. Furthermore, peripheral LPS injection markedly increased surface expression of IL-4 receptor-alpha (IL-4Rα), but not IL-10 receptor-1 (IL-10R1) on microglia. In BV-2 cells, IL-4, but not IL-10, re-directed LPS-activated microglia towards an M2 phenotype. Based on these findings, comparisons of M1 and M2 activation profiles, induction of IL-4Rα, and sensitivity to IL-4 were determined in microglia from adult (3-4 mo) and aged (18-22 mo) mice. In aged microglia, LPS promoted an exaggerated and prolonged M1 and M2 profile compared to adults. Moreover, IL-4Rα protein was not increased on aged microglia following LPS injection. To determine the consequence of impaired IL-4Rα upregulation, adult and aged mice were injected with LPS and activated microglia were then isolated and treated ex vivo with IL-4. While ex vivo IL-4 induced an M2 profile in activated microglia from adult mice, activated microglia from aged mice retained a prominent M1 profile. These data indicate that activated microglia from aged mice are less sensitive to the anti-inflammatory and M2-promoting effects of IL-4.

Immature immunosuppressive CD14+HLA-DR-/low cells in melanoma patients are Stat3hi and overexpress CD80, CD83, and DC-sign.

Myeloid-derived suppressor cells (MDSC) have emerged as key immune modulators in various tumor models and human malignancies, but their characteristics in humans remain to be unequivocally defined. In this study, we have examined circulating CD14(+)HLA-DR(-/low) MDSC in 34 advanced malignant melanoma (MM) patients. Their frequency is significantly increased and associated with disease activity. Contrary to the common notion that MDSC are a heterogeneous population of exclusively immature cells, we find the coexpression of markers associated with mature phenotype. We show for the first time the overexpression of CD80, CD83, and DC-Sign in human MDSC. Further, increased levels of signal transducer and activator of transcription 3 (Stat3), an important regulator in MDSC development and function, were noted in MM-MDSC. Stat3 was altered toward an active, phosphorylated state in the HLA-DR(-) population of CD14(+) cells and was more reactive to activating stimuli in patients. Importantly, inhibition of Stat3 abolished their suppressive activity almost completely. The described MM-MDSC use arginase in conjunction with other yet undefined mechanisms to suppress CD4(+) and CD8(+) T cells. Several observations suggest a redox imbalance in MDSC and indicate an important role of Stat3-dependent oxidative stress in MDSC-mediated T-cell suppression. These results emphasize the diversity of MDSC in human cancer and provide potential targets for therapeutic interventions.

[Expression of the human soluble interleukin 4 receptor in methylotropic yeast].

Interleukin-4 (IL-4) is a pleiotropic cytokine which plays a pivotal role in shaping immune responses. IL-4 mediates important proinflammatory functions in asthma, including the IgE isotype switch. IL-4 exerts its biological effects through binding to its receptor (IL-4R) complex, with the alpha chain as the high affinity binding subunit. Soluble IL-4R lacks the transmemberane and cytoplasmic domains, so it cannot induce cellular activation. By acting as a decoy to circulating IL-4 and neutralize its activity, its high specificity and affinity make it ideal as an IL-4 antagonist. Some companies have embarked the clinical research for asthma treatment with the sIL-4R and the result revealed well therapeutic effect. With RNA extracted from human monocyte as the template, The sIL-4R cDNA encoding the extracellular domain of IL-4R a chain was obtained by RT-PCR. Compared with the sIL-4R encoding sequence in GenBank, the nucleotide sequencing analysis indicated that there was a A-->G mutation at 148bp and the mutation caused Ile-->Val at 50th amino acid. According to the references, numerous polymorphisms have been identified in the IL-4R gene and the Ile50Val was the only known extracellular variant of human IL-4R. Then the recombinant vector pPIC9K/sIL-4R was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The recombinant sIL-4R was identified by SDS-PAGE, Western blot and Ligand binding blot. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed sIL-4R was about 30kD. And the Ligand binding blot analysis indicated the expressed sIL-4R had the biological activity. The sIL-4R, which had the biological activity, was successfully secretorily expressed in the Pichia pastoris (GS 115).