ALBERT

Description: We previously reported that monocyte adhesion to tumor necrosis factor-α (TNF-α)–treated endothelial cells increased expression of tissue factor and CD36 on monocytes. Using immunological cross-linking to mimic receptor engagement by natural ligands, we now show that CD15 (Lewis X), a monocyte counter-receptor for endothelial selectins may participate in this response. We used cytokine production as a readout for monocyte activation and found that CD15 cross-linking induced TNF-α release from peripheral blood monocytes and cells from the monocytic cell line MM6. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed an increase in steady-state TNF-α mRNA after 3 to 4 hours of cross-linking. CD15 cross-linking also concomitantly increased interleukin-1β (IL-1β) mRNA, while no apparent change was observed in the levels of β-actin mRNA, indicating specificity. To examine transcriptional regulation of cytokine genes by CD15 engagement, a CAT plasmid reporter construct containing IL-1β promoter/enhancer sequences was introduced into MM6. Subsequent cross-linking of CD15 increased CAT activity. CD15 engagement by monoclonal antibody also attenuated IL-1β transcript degradation, demonstrating that signaling via CD15 also had posttranscriptional effects. Nuclear extracts of anti-CD15 cross-linked cells demonstrated enhanced levels of the transcriptional factor activator protein-1, minimally changed nuclear factor-κB, and did not affect SV40 promoter specific protein-1. We conclude that engagement of CD15 on monocytes results in monocyte activation. In addition to its well-recognized adhesive role, CD15 may function as an important signaling molecule capable of initiating proinflammatory events in monocytes that come into contact with activated endothelium.

Description: Microarray analysis with 40 000 cDNA gene chip arrays determined differential gene expression profiles (GEPs) in CD34+ marrow cells from myelodysplastic syndrome (MDS) patients compared with healthy persons. Using focused bioinformatics analyses, we found 1175 genes significantly differentially expressed by MDS versus normal, requiring a minimum of 39 genes to separately classify these patients. Major GEP differences were demonstrated between healthy and MDS patients and between several MDS subgroups: (1) those whose disease remained stable and those who subsequently transformed (tMDS) to acute myeloid leukemia; (2) between del(5q) and other MDS patients. A 6-gene “poor risk” signature was defined, which was associated with acute myeloid leukemia transformation and provided additive prognostic information for International Prognostic Scoring System Intermediate-1 patients. Overexpression of genes generating ribosomal proteins and for other signaling pathways was demonstrated in the tMDS patients. Comparison of del(5q) with the remaining MDS patients showed 1924 differentially expressed genes, with underexpression of 1014 genes, 11 of which were within the 5q31-32 commonly deleted region. These data demonstrated (1) GEPs distinguishing MDS patients from healthy and between those with differing clinical outcomes (tMDS vs those whose disease remained stable) and cytogenetics [eg, del(5q)]; and (2) molecular criteria refining prognostic categorization and associated biologic processes in MDS.

Description: Gene expression profiles (GEPs) were obtained from marrow hematopoietic precursor cells (HPC)(CD34+ cells) from 30 myelodysplastic syndrome (MDS) patients: RARS 2, RA 15, RAEB 9, RAEBT 4; IPSS Low 11, Int-1 10, Int-2 5, High 4, and 6 Normal individuals. Fluorescently labeled cDNA was prepared from CD34+ cells (〉90% purity), isolated by immunomagnetic column separation, after reverse transcription of high fidelity PCR-amplified poly(A) RNA (aRNA). The Cy-conjugated nucleotides for aRNA were hybridized to 40,000 gene chip microarrays obtained from the Stanford Functional Genomics Microarray Facility. aRNA from pooled normal CD34+ marrow cells was used as a Reference standard. High resolution scans were obtained to compile a dataset for each microarray, through files submitted to the Stanford Microarray Database. Dendrograms generated by unsupervised hierarchical gene clustering indicated major differences of GEP between Normal and MDS patients. Significance Analysis for Microarray (SAM) yielded 2327 genes significantly differentially expressed by MDS vs Normal: 2269 genes overexpressed, 58 underexpressed, with a false positive rate of ~10%. Prediction Analysis of Microarray (PAM) distinctly separated the MDS and Normal patients, requiring a minimum of 31 genes (which were also SAM significant). Class analysis by PAM correctly predicted 29 of the 30 to be MDS and 5 of the 6 to be Normal. Four disparate differential GEP regions in the dendrograms, comprising predominantly genes of differing functional categories provided signatures associated with differing MDS clinical subgroups. Nine of 10 patients with poor clinical outcomes were associated with a differing GEP signature than that which occurred in 14 of 20 patients with relatively good outcomes. Compared to the remainder of MDS patients, those with 5q- syndrome (n=5) had a differing GEP signature, with under-expression of 1018 genes, 11 of which were within the 5q31–32 CDS. Two of these genes (antioxidant protein1 and interferon regulatory factor1) have previously been proffered as candidate genes for this syndrome. Analysis of FACS-sorted highly purified marrow HPC subsets: CD34+38+ (late) and CD34+38- (early HPCs), indicated these ratios to be 4.3±2.1 (n=2) for MDS and 3.2±1.2 (n=12) for Normals. These findings suggest that the differing GEPs between the MDS and Normal CD34+ cells were not due to major differences in their proportions of CD38 cell subsets. SAM and PAM significant differential GEPs were noted between these cell subsets (also differing between MDS and Normal), indicating alteration of gene expression during differentiation. Wnt1 and β-catenin1 (genes involved in cell self-renewal) were over-expressed in both MDS CD38- and CD38+ cells compared to Normal. These data demonstrate: (1) molecular differences between MDS and Normal HPCs and within HPC subsets; (2) GEP signatures characterizing MDS patients with differing cytogenetic abnormalities (eg, 5q-) and clinical outcomes; (3) molecular criteria refining the prognostic categorization of MDS; and (4) gene expression data aiding characterization of the heterogeneous nature of this spectrum of diseases.

Description: We previously reported that monocyte adhesion to tumor necrosis factor-α (TNF-α)–treated endothelial cells increased expression of tissue factor and CD36 on monocytes. Using immunological cross-linking to mimic receptor engagement by natural ligands, we now show that CD15 (Lewis X), a monocyte counter-receptor for endothelial selectins may participate in this response. We used cytokine production as a readout for monocyte activation and found that CD15 cross-linking induced TNF-α release from peripheral blood monocytes and cells from the monocytic cell line MM6. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed an increase in steady-state TNF-α mRNA after 3 to 4 hours of cross-linking. CD15 cross-linking also concomitantly increased interleukin-1β (IL-1β) mRNA, while no apparent change was observed in the levels of β-actin mRNA, indicating specificity. To examine transcriptional regulation of cytokine genes by CD15 engagement, a CAT plasmid reporter construct containing IL-1β promoter/enhancer sequences was introduced into MM6. Subsequent cross-linking of CD15 increased CAT activity. CD15 engagement by monoclonal antibody also attenuated IL-1β transcript degradation, demonstrating that signaling via CD15 also had posttranscriptional effects. Nuclear extracts of anti-CD15 cross-linked cells demonstrated enhanced levels of the transcriptional factor activator protein-1, minimally changed nuclear factor-κB, and did not affect SV40 promoter specific protein-1. We conclude that engagement of CD15 on monocytes results in monocyte activation. In addition to its well-recognized adhesive role, CD15 may function as an important signaling molecule capable of initiating proinflammatory events in monocytes that come into contact with activated endothelium.

Description: The nuclear receptor ligand all-trans retinoic acid (ATRA) causes dramatic terminal differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients, but it is less active in other malignancies. However, downstream mediators of the effects of ATRA are not well understood. We used a cDNA microarray to search for ATRA-regulated genes in the APL cell line NB4 and found that ATRA regulated several members of the tumor necrosis factor (TNF) pathway. Here we show that TNF can synergize with ATRA to induce differentiation, showing monocytic characteristics more typical of differentiation mediated by TNF than by ATRA. ATRA and TNF can also induce differentiation of the non-APL cell line U937. Underlying this response was an increase in TNF-induced nuclear factor-κB (NF-κB) DNA binding within 2 hours in the presence of ATRA and activation of NF-κB DNA binding and transcriptional activity in response to ATRA alone within 48 hours of ATRA treatment. Furthermore, we found a synergistic induction of the NF-κB target genes BCL-3, Dif-2, and TNF receptor 2 (TNFR2) in response to the combination of TNF and ATRA. These genes have been previously shown to play a role in TNF signaling, and amplification of such genes may represent a mechanism whereby TNF and ATRA can act synergistically. We propose that ATRA can prime cancer cells for differentiation triggered by TNF and suggest that targeting the TNF pathway in combination with ATRA may represent a novel route to treat leukemias. (Blood. 2003;102:237-245)

Description: Both Toll-like receptor 4 (TLR4)– and MD-2–deficient mice succumb to otherwise nonfatal Gram-negative bacteria inocula, demonstrating the pivotal role played by these proteins in antibacterial defense in mammals. MD-2 is a soluble endogenous ligand for TLR4 and a receptor for lipopolysaccharide (LPS). LPS-bound MD-2 transmits an activating signal onto TLR4. In this report, we show that both recombinant and endogenous soluble MD-2 bind tightly to the surface of live Gram-negative bacteria. As a consequence, MD-2 enhances cellular activation, bacterial internalization, and intracellular killing, all in a TLR4-dependent manner. The enhanced internalization of MD-2–coated bacteria was not observed in macrophages expressing Lpsd, a signaling-incompetent mutant form of TLR4, suggesting that the enhanced phagocytosis observed is dependent on signal transduction. The data confirm the notion that soluble MD-2 is a genuine opsonin that enhances proinflammatory opsonophagocytosis by bridging live Gram-negative bacteria to the LPS transducing complex. The presented results extend our understanding of the role of the TLR4/MD-2 signaling axis in bacterial recognition by phagocytes.