Identification of serum interleukin-13 and interleukin-13 receptor subunit expressions: Rheumatoid arthritis-associated interstitial lung disease.

AIM OF THE WORK: To identify the role of serum IL-13, and its receptor subunit expressions as a serologic marker of rheumatoid arthritis (RA)-associated ILD (RA-ILD). PATIENTS AND METHODS: Fifty RA patients with ILD and 50 RA patients without ILD were examined, in addition to 50 controls. Disease Activity Score in 28 joints (DAS-28), the Health Assessment Questionnaire (HAQ), and medication history were evaluated. ESR, CRP, RF, Anti-CCP, Serum Krebs von den Lungen-6 (KL-6), surfactant protein D (SP-D) levels, Interleukin 13 and its receptors (IL-13 Rα1 and L-13 Rα2), and mRNA relative expression levels in peripheral blood mononuclear cells (PBMCs) were measured. High-resolution computed tomography (HRCT) scores were used with all RA patients with interstitial lung disease. RESULTS: Mean age, percent of male affection, duration of the disease, DAS28 and MHAQ were significantly higher in the RA-ILD group than in the RA-no ILD group. ESR, CRP, RF, anti-CCP, serum KL-6, SP-D, IL-13 levels, IL-13 Rα1and IL-13 Rα2 mRNA expressions were significantly increased in RA patients compared to controls; in addition, their levels were significantly higher in the RA-ILD group than in the RA-no ILD group. Serum IL-13 levels and IL-13 Rα1and IL-13 Rα2 were positively correlated with RF, Anti-CCP, KL-6, SP-D, and the HRCT score (P < .001). CONCLUSIONS: Serum IL-13 and its receptor subunit expressions are useful biomarkers which can be used in detecting severity of the interstitial lung disease in RA patients.

IL-13R alpha 2 membrane and soluble isoforms differ in humans and mice.

Although mice have nanogram per milliliter serum levels of soluble (s) IL-13Ralpha2, humans lack sIL-13Ralpha2 in serum. Our data provide a mechanism for this biological divergence. In mice, discrete transcripts encoding soluble and membrane forms of IL-13Ralpha2 are generated by alternative splicing. We used small interfering RNA to specifically deplete the transcript encoding membrane (mem) IL-13Ralpha2 (full-length) or sIL-13Ralpha2 (DeltaEx10) in murine cells. Depletion of the full-length transcript decreased memIL-13Ralpha2 but had no effect on the level of sIL-13Ralpha2 in cell supernatants at baseline or following cytokine stimulation. Depletion of the DeltaEx10 transcript decreased sIL-13Ralpha2 in supernatants at baseline and following stimulation. In contrast to mice, we were unable to find a transcript encoding sIL-13Ralpha2 in humans and siRNA-mediated depletion of full-length IL-13Ralpha2 decreased both sIL-13Ralpha2 and memIL-13Ralpha2 in human cells. Inhibition of matrix metalloproteinases (MMP)/MMP-8 abolished production of sIL-13Ralpha2 from human cells. Thus, sIL-13Ralpha2 is derived exclusively from the memIL-13Ralpha2 transcript in humans through MMPs/MMP-8 cleavage of memIL-13Ralpha2, supporting a limited role for sIL-13Ralpha2 in humans and highlighting the potential importance of memIL-13Ralpha2 in human immunity. These observations require consideration when results of murine IL-13 studies are applied to humans.

[Roles of serum and urinary interleukins 13Ralpha2 and other cytokines in pediatric Henoch-Schonlein purpura].

OBJECTIVE: To study the roles of serum and urinary interleukins (IL)-13Ralpha2, IL-4, IL-6, IL-8 and tumor necrosis factor-alpha(TNF-alpha) in pediatric Henoch-Schonlein purpura (HSP). METHODS: Serum and urinary levels of IL-13Ralpha2, IL-4, IL-6, IL-8 and TNF-alpha were examined using ELISA in 52 children with HSP and 45 healthy children. The results were compared between the two groups. RESULTS: Serum levels of IL-13Ralpha2, IL-4, IL-6, IL-8 and TNF-alpha in HSP patients with or without renal lesions were higher than those in the control group (p<0.01 or 0.05). Urinary levels of IL-6 and TNF-alpha in HSP patients without renal lesions were higher than those in the control group (p<0.05). Except for urinary levels of IL-6 and TNF-alpha, urinary IL-13Ralpha2 levels in HSP patients with renal lesions (HSPN) were higher than those in the control group (p<0.05). CONCLUSIONS: Cytokines IL-13Ralpha2, IL-4, IL-6, IL-8 and TNF-alpha may play roles in the pathogenesis of pediatric HSP/HSPN.

Matrix metalloproteinase 8 contributes to solubilization of IL-13 receptor alpha2 in vivo.

BACKGROUND: IL-13 receptor alpha2 (IL-13R alpha 2) is a high-affinity receptor for IL-13, a central mediator of allergic asthma. It acts predominantly as a decoy receptor but can also contribute to IL-13 responses under certain conditions. IL-13R alpha 2 exists in soluble and membrane forms, which can both bind IL-13 and modulate its activity. Yet the proteolytic processes that contribute to the generation of soluble IL-13R alpha 2 are largely unknown. OBJECTIVE: We sought to investigate the role of matrix metalloproteinases (MMPs) in the generation of soluble IL-13R alpha 2. METHODS: Acellular cleavage assays by MMPs were performed by using glutathione-S-transferase fusion proteins of murine or human IL-13R alpha 2. IL-13R alpha 2 stable-transfected cells were used for analysis of surface expression and release of soluble IL-13R alpha 2. Wild-type and MMP-8-deficient mice were used for analysis of allergen-induced airway hyperresponsiveness and solubilization of IL-13R alpha 2. RESULTS: Among several MMPs tested, only MMP-8 cleaved IL-13R alpha 2. Treatment of transfected human or murine cells expressing high levels of surface IL-13R alpha 2 with MMP-8 resulted in release of soluble IL-13R alpha 2 into the supernatants, with a concomitant decrease in surface IL-13R alpha 2 levels. The IL-13R alpha 2 solubilized by MMP-8 retained IL-13 binding activity. In an asthma model MMP-8-deficient mice displayed increased airway hyperresponsiveness and decreased soluble IL-13R alpha 2 protein levels in bronchoalveolar lavage fluid compared with those seen in wild-type mice after house dust mite challenge. CONCLUSION: MMP-8 cleaves IL-13R alpha 2 in vitro and contributes to the solubilization of IL-13R alpha 2 in vivo.

A novel and sensitive ELISA reveals that the soluble form of IL-13R-alpha2 is not expressed in plasma of healthy or asthmatic subjects.

BACKGROUND: IL-13 plays a key regulatory role in asthmatic responses and immunity to parasitic infection. In vivo, IL-13R-alpha2 is a critical modulator of IL-13 bioactivity. When inducibly expressed on the surface of fibroblasts and other cell types under inflammatory conditions, IL-13R-alpha2 contributes to resolution of IL-13 responses. A soluble form of IL-13R-alpha2 (sIL-13R-alpha2) can be detected in murine circulation, and functions as a regulator of IL-13 bioactivity. In humans, sIL-13R-alpha2 has been more difficult to detect. Recently, novel assay systems have been described to quantitate sIL-13R-alpha2 in human circulation, and revealed unexpectedly high levels of sIL-13R-alpha2 in healthy subjects. OBJECTIVE: To verify sIL-13R-alpha2 quantitation in human plasma samples under stringent conditions of signal verification and false-positive detection. METHODS: A standard ELISA protocol was evaluated for specificity using false-positive detection reagents. A more stringent ELISA protocol was developed by optimizing the composition of blocking and dilution buffers. RESULTS: Using the stringent assay protocol, endogenous sIL-13R-alpha2 was undetectable in plasma samples from a total of 120 asthmatics and 20 healthy subjects, and in bronchoalveolar lavage fluid from 10 asthmatics and eight healthy subjects undergoing allergen challenge. CONCLUSION: These results underscore the necessity to perform rigorous assay controls in the biological matrix to be tested. Because the soluble form could not be demonstrated, our findings question a role for sIL-13R-alpha2 in the regulation of IL-13 bioactivity, and highlight the potentially important contribution of the membrane-bound form of IL-13R-alpha2 in humans.