The Combined CXCR1/CXCR2 Antagonist CXCL8(3-72) K11R/G31P Blocks Neutrophil Infiltration And Pulmonary Pathology In Guinea Pig Challenged By Klebsiella Pneumoniae

Objective:ELR(Glu-Leu-Arg)-CXC chemokines are important in acute responses to bacterial infections, wherein neutrophils are often critical to pathogen clearance. However, excessive neutrophil recruitment augments the pathology of many diseases. ELR+CXC chemokines bind CXCR1 and/or CXCR2 with a high affinity and have a potent chemotactic effect, particularly on neutrophils. CXCR1 and CXCR2 are coordinately expressed on the leukocytes. However, CXCR1 exhibits a slightly lower affinity for CXCL8 in vitro and requires more CXCL8 for internalization and recycling than CXCR2. Moreover, synthesis and release of CXCL8 can be induced by bacteria, bacterial products [e.g., lipopolysaccharide (LPS)]. We have developed a broad-spectrum ELR+CXC chemokine antagonist, CXCL8(3-72)K11R/G31P. In vitro, it effectively blocks neutrophil responses to CXCR1-and CXCR2-specific agonists, and ablates neutrophil responses to all mediators present in clinical bacterial pneumonia lesions. Klebsiella pneumoniae (Kp), accounts for a significant proportion of hospital-acquired urinary tract infections, pneumonia, septicemias, and soft tissue infections. Therefore, in this experiment we are trying to find out whether G31P also works very well in a guinea pig model of acute lung inflammation induced by the Klebsiella pneumoniae.Methods:For the preparation of the bacteria, we chose the optimal growth stage by drawing the time-growth curve of bacterium and diluted it with sterile saline. Then guinea pig were challenged with 0.2ml (10*9 cfu /ml) of Klebsiella pneumoniae through transtracheal injection. Meanwhile, administrated G31P(500ug/kg) or the same volume sterile saline as positive control by subcutaneously injection on the back. Also established antibiotic and hormones treated group. The guinea pig were sacrificed 24h later. Then count the number of neutrophils, assay the release of myeloperoxidase(MPO) in both lung tissue and the bronchoalveolar lavage fluid(BALF), observate the appearance of lung tissue and lung histopathology respectively. The RT-PCR was used to assay the expression of CXCL8 (IL-8), TNF and CXCL1 (IL-1) with a commercial one-step master mix kit.Results:The results show that, compared with the control group, the number of neutrophils decreased significantly, the secretion of MPO in both lung tissue and BALF dropped in G31P group, and there are a lot of differences in histopathology between G31P treated group and control.The mRNA level of inflammatory cytokines in G31P treated group were lower than that of control. The differences also happened between. G31P treated group and antibiotic, hormones groups.Conclusion:The combined CXCR1/CXCR2 antagonism-G31P had a significant therapeutic effect on guinea pig pulmonary inflammation and hemorrhage induced by Kp. In a word, G31P can partly block netrophil influx and tissue hemorrhage so as to exert a certain effect on this pneumonia model. Compared with antibiotic and hormones group, G31P treated group showed more effective.