Significant decreased lipopolysaccharide (LPS)-activated cytokine/receptor, chemokine, immunoregulatory, transcription, apoptotic, signaling and cell structure gene expression in cord blood (CB) compared to adult peripheral blood (APB) monocytes by oligonucleotide gene expression microarray: insight into differential apb versus CB monocyte structure and function

Abstract

Umbilical cord blood hematopoiesis and cellular immunity are developmentally immature when compared with adult peripheral blood (Cairo et al, Blood 90:4465,1997). We and others have demonstrated that umbilical cord blood transplantation (UCBT) is associated with decreased (grade III-IV) aGVHD, but delayed immune reconstitution (Abu-Ghosh/Cairo et al, BMT 24:535,1999). Recently, the contribution of allograft monocytes to the induction of GVHD and/or immune tolerance has been demonstrated (Aranha et al, Haematologica, 87:219, 2002). Monocytes are critically important in the generation of cytokines, chemokines, immune mediators and the initiation of the cellular immune responses. We and others have previously demonstrated significant dysregulated cytokine gene expression and protein production and in vitro functional activities of activated CB versus APB MNC. In this study we compared, by oligonucleotide microarray, the differential gene expression profiles of basal and LPS-activated APB versus CB monocytes. Briefly, Mo were purified from fresh CB or APB (N = 5) and stimulated with LPS (10 μg/ml, 18 hours). mRNA was isolated, reverse transcripted to cDNA, labeled and hybridized to oligonucleotides (Affymetrix, U95A) (Huang et al, Science 294:870, 2001). Data was analyzed by Microarray Suite Version 5.0 (Affymetrix) and hierarchical clustering analysis was performed by GeneSpring 5.0 software (Silicon Genetics). Quantitative real time PCR (LightCycler-RNA amplification SYBR Green I kit, Roche Molecular Biochemicals) was used to examine selected genes to confirm expression levels. We demonstrated patterns of significant differential amplified gene expression in LPS-activated APB versus CB monocytes including cytokine (G-CSF, 14 fold), chemokine (MIP-1α, 5 fold), immunoregulatory (MHC DRB1, 5 fold), transcription factor (JunB, 4 fold), signal transduction (STAT4, 5 fold), apoptotic regulation (BAX, 5 fold) and cell structure (ladinin 1, 6 fold) among others. These results suggest that some of these differentially expressed genes may in part be responsible for the differences in the incidence and severity of aGVHD, immunoreconstitution and/or the ability to cross more disparate HLA barriers following UCBT. Moreover, these data provide insight into the molecular basis for differential cellular function and immune responses between activated CB and APB monocytes. Umbilical cord blood hematopoiesis and cellular immunity are developmentally immature when compared with adult peripheral blood (Cairo et al, Blood 90:4465,1997). We and others have demonstrated that umbilical cord blood transplantation (UCBT) is associated with decreased (grade III-IV) aGVHD, but delayed immune reconstitution (Abu-Ghosh/Cairo et al, BMT 24:535,1999). Recently, the contribution of allograft monocytes to the induction of GVHD and/or immune tolerance has been demonstrated (Aranha et al, Haematologica, 87:219, 2002). Monocytes are critically important in the generation of cytokines, chemokines, immune mediators and the initiation of the cellular immune responses. We and others have previously demonstrated significant dysregulated cytokine gene expression and protein production and in vitro functional activities of activated CB versus APB MNC. In this study we compared, by oligonucleotide microarray, the differential gene expression profiles of basal and LPS-activated APB versus CB monocytes. Briefly, Mo were purified from fresh CB or APB (N = 5) and stimulated with LPS (10 μg/ml, 18 hours). mRNA was isolated, reverse transcripted to cDNA, labeled and hybridized to oligonucleotides (Affymetrix, U95A) (Huang et al, Science 294:870, 2001). Data was analyzed by Microarray Suite Version 5.0 (Affymetrix) and hierarchical clustering analysis was performed by GeneSpring 5.0 software (Silicon Genetics). Quantitative real time PCR (LightCycler-RNA amplification SYBR Green I kit, Roche Molecular Biochemicals) was used to examine selected genes to confirm expression levels. We demonstrated patterns of significant differential amplified gene expression in LPS-activated APB versus CB monocytes including cytokine (G-CSF, 14 fold), chemokine (MIP-1α, 5 fold), immunoregulatory (MHC DRB1, 5 fold), transcription factor (JunB, 4 fold), signal transduction (STAT4, 5 fold), apoptotic regulation (BAX, 5 fold) and cell structure (ladinin 1, 6 fold) among others. These results suggest that some of these differentially expressed genes may in part be responsible for the differences in the incidence and severity of aGVHD, immunoreconstitution and/or the ability to cross more disparate HLA barriers following UCBT. Moreover, these data provide insight into the molecular basis for differential cellular function and immune responses between activated CB and APB monocytes.