Dominant inflammatory cytokines might be different depending on the underlying causes of acute lung injury (ALI). The role of kertinocyte-derived chemokine (KC), a potent chemoattractant for neutrophils, has not been clearly established in hemorrhage-induced ALI. In this study, lung injury and cytokine expressison were evaluated in LPS- or hemorrhage-induced ALI models of BALB/c mice. The myeloperoxidase activities at 4 hr after hemorrhage and LPS-injection were 47.4+/-13.0 and 56.5+/-16.4 U/g, respectively. NF-kappa B activity peaked at 4 hr after hemorrhage, which was suppressed to the control level by anti-high mobility group B1 (HMGB1) antibody. Lung expressions of TNF-alpha, MIP-2, and IL-1beta were increased by LPS injection. However, there was only a minimal increase in IL-1beta and no expressions of TNF-alpha or MIP-2 in hemorrhage-induced ALI. In contrast, lung KC increased significantly at 4 hr after hemorrhage compared to control levels (83.1+/-12.3 vs. 14.2+/-1.6 pg/mL/mg by ELISA) (P<0.05). By immunohistochemical staining, lung neutrophils stained positive for KC. Increased KC was also observed in bronchoalveolar lavage fluid and plasma. KC plays an important role in hemorrhage-induced ALI.
Acute Lung Injury/etiology/*metabolism ; Animals ; Antibodies/immunology/metabolism ; Chemokine CXCL2/analysis ; Chemokines/analysis/blood/*physiology ; Chickens ; HMGB1 Protein/metabolism ; Humans ; Interleukin-1beta/analysis ; Lipopolysaccharides/toxicity ; Mice ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Neutrophils/immunology/metabolism ; Peroxidase/analysis ; Shock, Hemorrhagic/*complications ; Time Factors ; Tumor Necrosis Factor-alpha/analysis

OBJECTIVETo study the expression of hHSF in E. coli and its effect on the mobilization of hematopoietic stem/progenitor cells.

METHODSThe hHSF gene was obtained by overlapping PCR and cloned into the vector pET30a to yield pET30a-hHSF, which was transformed into E. coli BL21(DE3) and expressed with IPTG induction. Subsequently, rhHSF was purified by gel filtration and cation exchange chromatography and subjected to refolding. Molecular weight of hHSF was measured by MALDI-TOF Mass Spectroscopy. The N terminal amino acid sequence rhHSF was determined by protein sequencing. rhHSF was profiled in rhesus monkey for mobilization of peripheral blood stem cells. Eight rhesus monkeys were equally divided into two groups. The first group was administered single subcutaneous injection of 500 microg/kg hHSF, while the other one was administered 10 microg.kg(-1).d(-1) G-CSF for 4 days followed by a single subcutaneous injection of 500 microg/kg rhHSF.

RESULTSThe sequence coding hHSF was confirmed by sequencing and the induced-expression level was about 30% of total cell proteins. The purity of target protein was over 95%. The sequence of N terminal 10 amino acids and the amino acid composition were consistent with the theoretical parameters; molecular weight of rhHSF was 7540. The peripheral CD34(+) cells, CFU-GM yields, and neutrophils peaked at 3 h (16.3-folds increase compared with baseline), 1 h (1.9-folds increase) and 45 min (4.4-folds increase) respectively after the single injection of rhHSF. The addition of rhHSF after the last dose of G-CSF boosted these levels to 25.8-folds, 8.7-folds and 8.3-folds respectively.

CONCLUSIONhHSF is highly expressed in E. coli and rapidly mobilizes the hematopoietic stem/progenitor cells and neutrophils in rhesus monkeys. hHSF shows distinct synergistic effect with G-CSF.

Animals ; Chemokine CXCL2 ; chemistry ; genetics ; pharmacology ; Escherichia coli ; genetics ; Female ; Genetic Vectors ; genetics ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; methods ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Macaca mulatta ; Male ; Protein Folding ; Recombinant Proteins ; chemistry ; pharmacology ; Sequence Analysis, Protein ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization