| Acting as effector molecules of innate immunity, the importance of antimicrobial peptides in anti-infection are being reconstructed .People look forward to antimicrobial peptides being new antibiotics which can resist drug-fast micro-organism. Recently, a large body of research has suggested that besides antimicrobial activity, endogenetic antimicrobial peptides perform many activities including anti-infection, inflammatory reaction and wound healing. A role for the peptides in antiviral and immunomodulatory functions has recently been claimed. In human being, the most important researches in recent years are about defensins family and cathelicidin family. The antimicrobial peptide hCAP-18/LL-37 is a single cathelicidin defense peptide of human origin. Cathelicidins are so far only found in mammals and contain a conserved pro region of the cathelin type and a C-terminal variant domain, which is liberated by processing enzyme as a mature antimicrobial peptide. In humans the gene encoding hCAP-18/LL-37 is expressed in various tissues and cells but most pronounced in polymorphonuclear granulocytes and epithelial cells. Epithelial cells are constantly exposed to bacteria and phagocytes are recruited to sites of bacterial infections to terminate the bacterial spread and eliminate the invader. Their antimicrobial spectrum covers gram-positive and -negative bacteria as well as fungi and certain viruses. Thus, hCAP-18/LL-37 is an effector in the armament of immediate and early host defenses. In addition to bactericidal activity, hCAP-18/LL-37 exhibits chemotactic activity for several types of inflammatory and immune cells. Thus hCAP-18/LL-37 exhibits a dual adaptive roles, being involved in killing bacteria and additionally in recruiting host effector cells for enhancing early innate defenses and for initiating the adaptive axis of immunity and modulate adaptive immunity.However the exact mechanisms of interaction between innate immunity and adaptive immunity have not been elucidated. In an attempt to unravel the mechanism of hCAP-18/LL-37 on modulating adaptive immunity, we have the following works:Part I. Gene cloning and eukaryotic expression of hCAP-18/LL-371. Detect the gene expression of hCAP-18/LL-37 in some kinds of cellsThe gene expression of hCAP-18/LL-37 was determined by reverse-transcription-polymerase chain reaction. The cells being detected were heterophil granulocyte from healthy human PBMC, normal human cell lines HEK293, FL and human tumor cell lines Hela, MCF-7, SMMC-7721, A549 and BGC-823. The results demonstrate that the gene expression of hCAP-18/LL-37 in was detected and not detected in cell lines HEK293, FL, Hela, MCF-7, SMMC-7721, A549 and BGC-823.2. Gene cloning of hCAP-18/LL-37 from heterophil granulocyte from healthy human PBMC The primer designed basing on the gene sequence accessing in genebank, including the genes coding the full cDNA and of mature peptide. Reverse-transcription-polymerase chain reaction was used to obtain the gene fragment. We ligated the two fragments into the vector PMD18-T. Identifying by restriction enzyme confirmed that we had obtain the genes coding the full cDNA and of mature peptide.3. Construct the eukaryotic expression vector of hCAP-18/LL-37We constructed two recombinant eukaryotic expression vector including the genes coding the full cDNA and mature peptide. They are pcDNA4 /Myc-His-hCAP-18 and pcDNA4/Myc-His-LL-37. Identifying by restriction enzyme and DNA sequencing it was confirmed that we have constructed two recombinant eukaryotic expression vector successfully. Then we transfected transiently them into the Hela cell lines and HEK293 cell lines. Identifying by RT-PCR and Western Blot, the expression of hCAP -18/LL-37 were detected in cells and supernatant.4. Establishment of stable transfected Hela cell linesWe transfected pcDNA4/Myc-His-hCAP-18 and pcDNA4/Myc-His-LL -37 into Hela cell lines and screened the positive clones. We obtained two cell lines of stable transfection for pcDNA4/Myc-His-hCAP-18 and three cell lines of stable transfection for pcDNA4/Myc-His-LL -37. Identifying by RT-PCR and Western Blot , the expression of hCAP -18/LL-37 were detected in cells and supernatant.Part II. The effects on differentiation and maturation of dendritic cells by hCAP-18/LL-37To study the effects on maturation and functions of dendritic cells by hCAP-18/LL-37, first we separated the PBMC from healthy human peripheral blood and obtain the precursor cells of DC by adherence. Then we cultured them in RPMI1640 medium containing rhIL-4 and rhGM-GSF for 7 days and obtained immature DCs. Then we divided the DCs into 8 groups: the group of DC stimulated by supernatant of untransfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected Hela cells, the group of DC stimulated by supernatant of untransfected HEK293 cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected HEK293 cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected HEK293 cells and the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected HEK293 cells. We collected DCs after being cultured with the 8 kinds of supernatant.To investigate the maturation of DC stimulated by hCAP-18/ LL-37, we detected the surface marker on DCs by FCM. The results showed that CD83 was not up-regulated which compared between the group of DC stimulated by supernatant of untransfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected Hela cells, the group of DC stimulated by supernatant of untransfected HEK293 cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected HEK293 cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected HEK293 cells and the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected HEK293 cells. CD86, CD40 and HLA-DR was up-regulated significantly compared between the group of DC stimulated by supernatant of untransfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected Hela cells, the group of DC stimulated by supernatant of untransfected HEK293 cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected HEK293 cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected HEK293 cells and the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected HEK293 cells. These findings suggest that hCAP-18/LL-37 can up-regulate the expression of DC surface marker CD86, CD40 and HLA-DR and promote DC maturation and antigen presenting.To observe the cell cycle and apoptosis of DC influenced by hCAP-18/ LL-37, we detected it by FCM. The results showed that in the group of DC stimulated by supernatant of untransfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected Hela cells, the percent of total S phase are 25.76 %,31.11 %,30.38 % and 31.37 %, the percent of apoptosis are 0.12 %,0.69 %,0.55 % and 0.27%. The group of DC stimulated by supernatant of untransfected HEK293 cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected HEK293 cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected HEK293 cells and the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected HEK293 cells, the percent of total S phase are 32.30 %,31.89 %,35.74 % and 34.29%, the percent of apoptosis are 0.40%,0.11%,0.28% and 1.31%. These findings suggest that hCAP-18/LL-37 can not induce apoptosis of DC.To observe the ability of stimulating allogenic T lymphocyte of DC influenced by hCAP-18/LL-37, we performed mixtured lymphocyte reaction. The results showed that it had significance between the group of DC stimulated by supernatant of untransfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected Hela cells and the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected Hela cells (P<0.01). But it had no significance between the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected Hela cells and the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected Hela cells(P>0.05). Similar results were obtained in HEK293. These findings suggest that hCAP-18/ LL-37 can promote the ability of stimulating T cells responses by DC.To observe the ability of stimulating T lymphocytes transformation from human PBMC by hCAP-18/ LL-37, we used MTT assay to detect the transformation ability of T cells. The results showed that it had significance between the group of DC stimulated by supernatant of untransfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His vector transfected Hela cells and the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected Hela cells, the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected Hela cells (P<0.01). But it had no significance between the group of DC stimulated by supernatant of pcDNA4/Myc-His-hCAP-18 transfected Hela cells and the group of DC stimulated by supernatant of pcDNA4/Myc-His-LL-37 transfected Hela cells(P>0.05). Similar results were obtained in HEK293. These findings suggest that hCAP-18/ LL-37 can promote the transformation ability of stimulating T cells.Taken together, this study based on gene engineering, cloned the hCAP-18/LL-37 gene and constructed the recombinant eukaryotic expression vector for full-lenth peptide and mature peptide. We transfected them into cell lines and established the stable transfected cell lines. Identifying by RT-PCR and Western Blot, hCAP-18/LL-37 expression was detected in transfected cells and supernatant. To study the effects on maturation and functions of dendritic cells by hCAP-18/LL-37, first we separated the PBMC from healthy human peripheral blood and obtain the precursor cells of DC by adherence. The results showed that hCAP-18/LL-37 can up-regulate the expression of DC surface marker CD86, CD40 and HLA-DR. hCAP-18/LL-37 also can promote the ability of stimulating T cells responses by DC and the transformation ability of stimulating T cells.These findings suggest that hCAP-18/LL-37 can promote maturation and ability of antigen presentation of DC to participate the adaptive immunity. These researches can provide some theory proof for application of hCAP-18/LL-37 on anti-infection and immunomodulation.
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