Pulmonary immune cells and inflammatory cytokine dysregulation are associated with mortality of IL-1R1 -/-mice infected with influenza virus (H1N1).

Respirovirus infection can cause viral pneumonia and acute lung injury (ALI). The interleukin-1 (IL-1) family consists of proinflammatory cytokines that play essential roles in regulating immune and inflammatory responses in vivo. IL-1 signaling is associated with protection against respiratory influenza virus infection by mediation of the pulmonary anti-viral immune response and inflammation. We analyzed the infiltration lung immune leukocytes and cytokines that contribute to inflammatory lung pathology and mortality of fatal H1N1 virus-infected IL-1 receptor 1 (IL-1R1) deficient mice. Results showed that early innate immune cells and cytokine/chemokine dysregulation were observed with significantly decreased neutrophil infiltration and IL-6, TNF-α, G-CSF, KC, and MIP-2 cytokine levels in the bronchoalveolar lavage fluid of infected IL-1R1 -/- mice in comparison with that of wild type infected mice. The adaptive immune response against the H1N1 virus in IL-1R1 -/- mice was impaired with downregulated anti-viral Th1 cell, CD8+ cell, and antibody functions, which contributes to attenuated viral clearance. Histological analysis revealed reduced lung inflammation during early infection but severe lung pathology in late infection in IL-1R1 -/- mice compared with that in WT infected mice. Moreover, the infected IL-1R1 -/- mice showed markedly reduced neutrophil generation in bone marrow and neutrophil recruitment to the inflamed lung. Together, these results suggest that IL-1 signaling is associated with pulmonary anti-influenza immune response and inflammatory lung injury, particularly via the influence on neutrophil mobilization and inflammatory cytokine/chemokine production.

Molecular Cloning of Human Interleukin-5 cDNA and Its Expression in E. coli.

hIL-5 cDNA was amplified through reverse transcription-polymerase chain reaction from peripheral blood lymphocytes induced with PMA and calcium ionophore A23187. The hIL-5 fragment was cloned into pUC18 and its sequence was identified to be hIL-5 cDNA sequence. The fragment which encodes hIL-5 mature polypeptide was amplified and cloned into pBV220 to express hIL-5 recombinant protein in E. coli, but there was no expected recombinant protein expressed. Only one clone expressed a 10kD peptide at high level. DNA sequencing showed that this clone had an adenosine deletion at 86th codon in hIL-5 cDNA and expressed a polypeptide of 93 amino acids at high level, and hIL-5 cDNA had not yet been expressed in E.coli successfully. These results suggested that two consecutive rare codons after 86th codon in hIL-5 cDNA could block polypeptide synthesis in E. coli. Through recombinant PCR technology the rare codons after 86th codon in hIL-5 cDNA were mutated into corresponding codons preferentially used in E. coli, and the mutated hIL-5 cDNA was highly expressed in pBVhIL5-H/DH5alpha to approximately 15% TCP after thermal induction. hIL-5 recombinant protein existed as inclusion bodies in E. coli. ELISA with a cross-reacting rat anti-mIL-5 monoclonal antibody confirmed the expressed product was hIL-5 recombinant protein.