Human Peripheral Blood CD4 Research Articles

Different rates of telomere shortening and telomerase activity reduction in CD8 T and CD16 NK lymphocytes with ageing

Telomeres are specialised structures located at the end of eukaryotic chromosomes, that get short during progressive cell divisions. Therefore, telomere may be an indicator of the mitotic history of a cell and it is also a determining factor for the residual cell life span. One mechanism, compensating for the telomere erosion, involves the induction of telomerase, a ribonucleoprotein-enzyme able to synthesize telomeric DNA repeats. In this study, old subjects of two consecutive decades were compared with a group of young controls to investigate whether ageing-related modifications differently affects telomere length and telomerase activity of human peripheral blood CD8 T and CD16 NK lymphocytes. Telomeres in individual cells were measured by flow-FISH and telomerase activity was determined using the TeloTAGGG telomerase PCR ELISA PLUS kit. Both CD8 T and NK lymphocytes showed an age-associated loss of telomeres at rates that were different between the subsets together with an age-associated reduction of telomerase activity that was progressive in CD8 and late in NK lymphocytes. We can assume that preserved innate immune response in the elderly is due to the negligible telomere shortening and the maintained telomerase expression that could allow NK cells of octogenarians to delay replicative senescence.

Detection of a functional hybrid receptor gammac/GM-CSFRbeta in human hematopoietic CD34+ cells.

A functional hybrid receptor associating the common γ chain (γc) with the granulocyte/macrophage colony-stimulating factor receptor β (GM-CSFRβ) chain is found in mobilized human peripheral blood (MPB) CD34+ hematopoietic progenitors, SCF/Flt3-L primed cord blood (CB) precursors (CBPr CD34+/CD56−), and CD34+ myeloid cell lines, but not in normal natural killer (NK) cells, the cytolytic NK-L cell line or nonhematopoietic cells. We demonstrated, using CD34+ TF1β cells, which express an interleukin (IL)-15Rα/β/γc receptor, that within the hybrid receptor, the GM-CSFRβ chain inhibits the IL-15–triggered γc/JAK3-specific signaling controlling TF1β cell proliferation. However, the γc chain is part of a functional GM-CSFR, activating GM-CSF–dependent STAT5 nuclear translocation and the proliferation of TF1β cells. The hybrid receptor is functional in normal hematopoietic progenitors in which both subunits control STAT5 activation. Finally, the parental TF1 cell line, which lacks the IL-15Rβ chain, nevertheless expresses both a functional hybrid receptor that controls JAK3 phosphorylation and a novel IL-15α/γc/TRAF2 complex that triggers nuclear factor κB activation. The lineage-dependent distribution and function of these receptors suggest that they are involved in hematopoiesis because they modify transduction pathways that play a major role in the differentiation of hematopoietic progenitors.

Spontaneous and rapid reexpression of functional CXCR4 by human steady-state peripheral blood CD34+ cells.

Although only 5% of steady-state peripheral blood (PB) CD34+ cells were found to express chemokine receptor CXCR4, 45% of the cells became CXCR4+ after incubation at 37 degrees C for 4 hours. In contrast, there were no remarkable differences between PB CD34+ cells before and after the 37 degrees C incubation in their expression of selectin ligand, VLA-4, and VLA-5 or in their affinity for VCAM-1 or fibronectin. This increase in CXCR4 expression level was inhibited by the addition of brefeldin A, actinomycin D, or cycloheximide. When PB CD34+ cells with CXCR4 expression levels enhanced by a 4-hour preincubation at 37 degrees C or bone marrow (BM) CD34+ cells were exposed overnight to stromal cell-derived factor 1 (SDF-1), the expression levels of CXCR4 were greatly reduced, and when SDF-1 was removed, CXCR4 levels were thereafter up-regulated. The reexpressed CXCR4 was able to elicit integrin-dependent migration of hematopoietic progenitor cells. There was no difference in the severe combined immunodeficient mouse repopulating cell activity between PB CD34+ cells with and cells without a 37 degrees C preincubation.

Skin-homing CLA+ T cells and regulatory CD25+ T cells represent major subsets of human peripheral blood memory T cells migrating in response to CCL1/I-309

Functionally distinct T cell subsets exhibit specific chemokine receptor profiles that regulate their tissue localization. Here, we show that human peripheral blood CD4(+) and CD8(+) cutaneous (CLA(+)), but not intestinal memory (integrin beta(7) (+)) nor IL-4-producing T cells, represent major subpopulations of circulating T cells that specifically migrate in response to the chemokine I-309/CCL1 by virtue of CCR8 expression. Expression of CCR8 is markedly up-regulated upon activation and in vitro culture of human CLA(+) T cells, suggesting the involvement of CCR8 in localization of cutaneous memory T cells to the skin. Interestingly, amongst circulating memory CD4(+)CD45RO(+) T cells, chemotactic responsiveness to CCL1 is restricted to cells expressing CD25 and/or CLA surface markers for regulatory T cells (Treg) and skin-homing T cells and maximal responsiveness is observed on CLA(+)CD25(+)T cells. Such pattern of CCL1 responsiveness suggests that the CCR8/CCL1 axis may regulate trafficking of cutaneous Treg and memory T cells into the skin.

Skin-homing CLA+ T cells and regulatory CD25+ T cells represent major subsets of human peripheral blood memory T cells migrating in response to CCL1/I-309.

Functionally distinct T cell subsets exhibit specific chemokine receptor profiles that regulate their tissue localization. Here, we show that human peripheral blood CD4(+) and CD8(+) cutaneous (CLA(+)), but not intestinal memory (integrin beta(7) (+)) nor IL-4-producing T cells, represent major subpopulations of circulating T cells that specifically migrate in response to the chemokine I-309/CCL1 by virtue of CCR8 expression. Expression of CCR8 is markedly up-regulated upon activation and in vitro culture of human CLA(+) T cells, suggesting the involvement of CCR8 in localization of cutaneous memory T cells to the skin. Interestingly, amongst circulating memory CD4(+)CD45RO(+) T cells, chemotactic responsiveness to CCL1 is restricted to cells expressing CD25 and/or CLA surface markers for regulatory T cells (Treg) and skin-homing T cells and maximal responsiveness is observed on CLA(+)CD25(+)T cells. Such pattern of CCL1 responsiveness suggests that the CCR8/CCL1 axis may regulate trafficking of cutaneous Treg and memory T cells into the skin.

Expression and affinity of homing-related molecules on steady-state adult and neonate human PB CD34+ cells and their SRC activity.

Although the vast majority of hematopoietic progenitor cells (HPCs) reside within the bone marrow (BM), a small number of HPCs also continuously circulate in the peripheral blood (PB). The examination of the fate of blood-borne HPCs in parabiotic mice, which are surgically conjoined and share a common circulation, recently revealed that steady-state PB HPCs play a physiological role in, at least, the functional re-engraftment of unconditioned BM. To assess the possibility that human HPCs have a similar function, in this study we examined the expression level and affinity of the homing-related molecules, as well as the SCID mouse reconstituting cell (SRC) activity of human PB CD34+ cells, and compared adults with neonates. There was no remarkable difference between adults and neonates in the expression of E- and/or P-selectin ligands by PB CD34+ cells or in these cells' affinity to VCAM-1. In contrast, the expression level of CXCR4 on PB CD34+ cells was much lower in adults than in neonates. Adult cells also showed a much lower SRC activity than neonates. These results suggest that human PB HPCs may contribute to steady-state hematopoiesis in the BM of neonates to some extent, but not so much in adults.

Extracellular adenine nucleotides inhibit the activation of human CD4+ T lymphocytes.

ATP has been reported to inhibit or stimulate lymphoid cell proliferation, depending on the origin of the cells. Agents that increase cAMP, such as PGE(2), inhibit human CD4(+) T cell activation. We demonstrate that several ATP derivatives increase cAMP in both freshly purified and activated human peripheral blood CD4(+) T cells. The rank order of potency of the various nucleotides was: adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) approximately 2'- and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP >> dATP, 2-propylthio-beta,gamma-dichloromethylene-D-ATP, UDP, UTP. This effect did not involve the activation of A(2)Rs by adenosine or the synthesis of prostaglandins. ATPgammaS had no effect on cytosolic calcium, whereas BzATP induced an influx of extracellular calcium. ATPgammaS and BzATP inhibited secretion of IL-2, IL-5, IL-10, and IFN-gamma; expression of CD25; and proliferation after activation of CD4(+) T cells by immobilized anti-CD3 and soluble anti-CD28 Abs, without increasing cell death. Taken together, our results suggest that extracellular adenine nucleotides inhibit CD4(+) T cell activation via an increase in cAMP mediated by an unidentified P2YR, which might thus constitute a new therapeutic target in immunosuppressive treatments.

CD8+CD28- T cells: certainties and uncertainties of a prevalent human T-cell subset.

Human peripheral blood CD8+ T cells comprise cells that are in different states of differentiation and under the control of complex homeostatic processes. In a number of situations ranging from chronic inflammatory conditions and infectious diseases to ageing, immunodeficiency, iron overload and heavy alcohol intake, major phenotypic changes, usually associated with an increase in CD8+ T cells lacking CD28 expression, take place. CD8+CD28- T cells are characterized by a low proliferative capacity to conventional stimulation in vitro and by morphological and functional features of activated/memory T cells. Although the nature of the signals that give origin to this T-cell subset is uncertain, growing evidence argues for the existence of an interplay between epithelial cells, molecules with the MHC-class I fold and CD8+ T cells. The possibility that the generation of CD8+CD28- T cells is the combination of TCR/CD3zeta- and regulatory factor-mediated signals as a result of the sensing of modifications of the internal environment is discussed.

Ex vivo expansion of stem and progenitor cells in co-culture of mobilized peripheral blood CD34+ cells on human endothelium transfected with adenovectors expressing thrombopoietin, c-kit ligand, and Flt-3 ligand.

To optimize conditions for ex vivo expansion of adult hematopoietic stem cells, we evaluated the co-culture of G-CSF mobilized human peripheral blood (PB) CD34(+) cells with endothelial cells engineered to overexpress various hematopoietic growth factors. Immortalized human bone marrow endothelial cells (BMEC) transfected with an expression vector carrying cDNA encoding the human telomerase reverse transcriptase (hTERT) and human umbilical vein endothelial cells (HUVEC) were transfected with combinations of adenovectors expressing murine c-kit ligand (KL), human thrombopoietin (TPO), human Flt3 ligand (FL), and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Ex vivo expansion of PB CD34(+) cells from normal donors and non-Hodgkin lymphoma (NHL) patients in endothelial co-culture was evaluated weekly for total cell production, progenitor (CFU-GM, BFU-E) cell production, and stem cell production as measured by Week-5 Cobblestone Area Forming Cell assay (Wk-5 CAFC). HUVEC transfected with adenovectors expressing TPO, KL, and FL provided the best co-culture system for expanding CD34(+) cells. Maximal total nuclear cell, CFU-GM, and Wk-5 CAFC production occurred between weeks 2 and 3 with 113-fold, 25-fold, and 2.2-5.5-fold expansions, respectively. We did not detect significant differences when GM-CSF was added to the co-culture system. Expansion was also obtained using recombinant human cytokines, but was not maintained beyond 3 weeks. We demonstrated that continuous generation of high levels of TPO, FL, and KL as well as other factors secreted by endothelium provided a clinically relevant co-culture method for ex vivo expansion of stem and progenitor cells from cryopreserved CD34(+) populations.

Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34(+) cells after ex vivo expansion.

The relationship between phenotype and function in ex vivo-cultured human hematopoietic stem cells (HSC) remains poorly understood. We investigated the effects of a short-term serum-free culture on the relationship between stem cell phenotype, cell division history, and function in human CD34(+) cells. G-CSF-mobilized peripheral CD34(+) cells were cultured for 4 days with stem cell factor, flt-3 ligand, and thrombopoietin. The phenotype (CD34, CD38, HLA-DR, c-kit), cell division history, colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and NOD/SCID repopulating activities were evaluated at Day 0 and 4. We observed a loss of CD38, HLA-DR, and c-kit surface expression resulting in a drastic increase in CD34(+)CD38(-), CD34(+)HLA-DR(-), and CD34(+)c-kit(-/low) cells at Day 4. In contrast, the frequency of Thy-1(+) cells was maintained. We observed a 1.3-fold expansion of CFC, a 4.8-fold increase in LTC-IC, and an overall maintenance of the NOD/SCID repopulating cell activity. CD34(+)CD38(-) and CD34(+)HLA-DR(-) cells detected at Day 4 displayed the most active pattern of division (4 to 5 divisions) whereas 60% of CD34(+)Thy-1(+) cells divided 0 to 2 times during the same period. At Day 4, the NOD/SCID repopulating activity was associated with Thy-1(+) cells with no more than 2 divisions. Our results show that the relationship between stem cell phenotype and function is dramatically altered in cultured CD34(+) cells. Thy-1 expression and cell division history appear to be superior to CD38, HLA-DR, and c-kit, or to homing molecules (CXCR4, VLA-4) as predictors of the repopulating activity of cultured peripheral CD34(+) cells.

Octamer proteins inhibit IL-4 gene transcription in normal human CD4 T cells.

The balance of Th1 (eg, interleukin-2 (IL-2)) and Th2 (eg, IL-4) cytokines produced by CD4 T cells markedly influences the outcome of the adaptive immune response. Although octamer transcription factor proteins increase IL-2 transcription in T cells, their role in IL-4 gene transcription remains controversial. We have previously shown and now confirm that the proximal octamer binding site of the human IL-4 promoter, which separates the two most proximal NFAT binding sites, is bound prior to, but not after, activation in vivo. Since these two NFAT sites are essential for optimal IL-4 promoter activity, this suggested that prior engagement by octamer proteins might prevent adjacent NFAT binding and inhibit IL-4 gene transcription. In support of this hypothesis, here we show that NFAT proteins are unable to bind to a combined octamer/NFAT site unless the octamer proteins are competed away. Moreover, activity of an IL-4 reporter gene mutated in the proximal octamer binding site is increased compared to the wild-type promoter in human peripheral blood CD4 T cells. In addition, over-expression of either Oct-1 or Oct-2 decreased wild-type IL-4 promoter activity, while increasing IL-2 promoter activity. No decrease in promoter activity was seen when Oct-1 or Oct-2 was over-expressed with the octamer-mutant IL-4 reporter gene. Thus, octamer proteins are candidates to promote a Th1 rather than Th2 pattern of cytokine gene expression by activated CD4 T cells.

Differential effects of autologous serum on CD34(+) or monocyte-derived dendritic cells.

Dendritic cells (DC) with potentially important clinical applications have been generated from human peripheral blood monocytes and CD34(+) cells in the presence of recombinant cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-4 (IL-4) and GM-CSF + tumor necrosis factor-alpha (TNF-alpha), respectively. Many of the studies generating DC have included fetal calf serum, which is not desirable due to the risk of immune reactions and infectious disease transmission. Additionally, low DC yields have been reported using serum-free media. In this study, we investigate supplementing serum-free media with autologous serum and plasma for DC generation from monocytes and CD34(+) cells. Our results show that functional DC can be reproducibly obtained in the presence of autologous serum using monocytes and CD34(+) cells as the starting populations. However, with the addition of autologous serum, a differential effect is observed in the phenotypic characterization of these culture-derived DC. Monocytes cultured for 7 days in X-VIVO 15 serum-free media in the presence of GM-CSF + IL-4 showed down-regulation of CD14 with increased expression of HLA-DR, mannose receptor, CD80, and CD86, along with highly up-regulated CD1a(+) expression. The addition of autologous serum to serum-free media in monocyte cultures resulted in a dose-dependent decrease in the CD1a(+) expression generating a distinct subset of CD1a(+/-) cells expressing HLA-DR, mannose receptor, CD80, and CD86. Upon stimulation with CD40L cells, both monocyte-derived DC subsets CD1a(+/-) and CD1a(++) were capable of maturation measured by CD83 and CD86 up-regulation. Data suggest the differences in the monocyte-derived DC in serum-free (CD1a(++)) or autologous serum (CD1a(+/-)) supplemented cultures is of a qualitative nature, rather than quantitative. CD1a(+) and CD14(+) cells expressing HLA-DR, mannose receptor, CD80, and CD86 were generated in 7 days from CD34(+) cells in serum-free media. A quantitative effect was obtained when cultures were supplemented with autologous serum, resulting in a significant enhancement of CD34-derived DC generated. These results demonstrate generation of DC from two different starting populations using serum-free media that can be enhanced with the addition of autologous serum. Interestingly, a differential effect was observed in the phenotypic characterization of these culture-derived DC.

HIV-protease inhibitors contribute to P-glycoprotein efflux function defect in peripheral blood lymphocytes from HIV-positive patients receiving HAART.

P-glycoprotein (P-gp) has been found expressed in normal human cells, such as bone marrow and peripheral blood cells. The aim of this study was to investigate whether HIV-protease inhibitors (HIV-PIs) interact with P-gp efflux function in normal human peripheral blood lymphocytes (PBLs) and CD34+ progenitor cells. Moreover, we analyzed the in vivo effect of HIV-PIs on P-gp function in PBLs from HIV-infected patients receiving highly active antiretroviral therapy (HAART). We found that HIV-PIs (i.e., ritonavir, saquinavir, nelfinavir and indinavir) interfere with P-gp function in normal PBLs as demonstrated by the reduced efflux of rhodamine 123 (Rh123). This effect was dose-dependent and suggested the following hierarchy: ritonavir > saquinavir > nelfinavir > indinavir. We further analyzed the effect of HIV-PIs on the P-gp function in specific PBLs subsets. Our results show an HIV-PI-induced inhibition of P-gp function in CD4+ and CD8+ T cell subsets, mostly caused by the effect on the naive compartment of both CD4+ and CD8+ T cells. The same inhibitory effect was found in CD34+ hematopoietic progenitor cells. With respect to the in vivo evaluation of P-gp function in PBLs from HIV-infected patients, we found reduced levels of Rh123 efflux that reached the lowest value in AIDS patients receiving HAART. We concluded that HIV-PIs interfere with P-gp function in major cellular targets for HIV infection, such as CD4+ T cells and CD34+ progenitor cells. This ability may contribute to P-gp efflux function defect found in HIV-infected patients and suggests that drug interaction studies are crucial to an overall understanding of the effects of this important group of drugs.

HIV-Protease Inhibitors Contribute to P-Glycoprotein Efflux Function Defect in Peripheral Blood Lymphocytes From HIV-Positive Patients Receiving HAART

Summary: P-glycoprotein (P-gp) has been found expressed in normal human cells, such as bone marrow and peripheral blood cells. The aim of this study was to investigate whether HIV-protease inhibitors (HIV-PIs) interact with P-gp efflux function in normal human peripheral blood lymphocytes (PBLs) and CD34+ progenitor cells. Moreover, we analyzed the in vivo effect of HIV-PIs on P-gp function in PBLs from HIV-infected patients receiving highly active antiretroviral therapy (HAART). We found that HIV-PIs (i.e., ritonavir, saquinavir, nelfinavir and indinavir) interfere with P-gp function in normal PBLs as demonstrated by the reduced efflux of rhodamine 123 (Rh123). This effect was dose-dependent and suggested the following hierarchy: ritonavir > saquinavir > nelfinavir > indinavir. We further analyzed the effect of HIV-PIs on the P-gp function in specific PBLs subsets. Our results show an HIV-PI-induced inhibition of P-gp function in CD4+ and CD8+ T cell subsets, mostly caused by the effect on the naive compartment of both CD4+ and CD8+ T cells. The same inhibitory effect was found in CD34+ hematopoietic progenitor cells. With respect to the in vivo evaluation of P-gp function in PBLs from HIV-infected patients, we found reduced levels of Rh123 efflux that reached the lowest value in AIDS patients receiving HAART. We concluded that HIV-PIs interfere with P-gp function in major cellular targets for HIV infection, such as CD4+ T cells and CD34+ progenitor cells. This ability may contribute to P-gp efflux function defect found in HIV-infected patients and suggests that drug interaction studies are crucial to an overall understanding of the effects of this important group of drugs.

Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation.

The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.

Induction of Telomerase Activity During Development of Human Mast Cells from Peripheral Blood CD34+ Cells: Comparisons with Tumor Mast-Cell Lines

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+ /CD117+ /CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+ /CD117+ /CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.

Further Characterization of FcγRII and FcγRIII Expression by Cultured Human Mast Cells

Background: We have reported that resting human mast cells exhibit minimal expression for FcγRI, and that interferon-γ will upregulate this expression. The expression of FcγRII and FcγRIII by human mast cells remains to be fully examined. Methods: To investigate FcγRII and FcγRIII expression, we determined mRNA and protein expression of FcγRII and FcγRIII in human peripheral blood CD34+ derived cultured mast cells by RT-PCR and flow cytometry. The expression of FcγRII and FcγRIII in intact and permeabilized mast cells was also compared. We measured histamine release to monitor mast cell degranulation following cross-linking of FcγRII. Results: We found by RT-PCR that resting human mast cells exhibit mRNA for FcγRIIA, FcγRIIb1, FcγRIIb2 and FcγRIII but not FcγRIIC. FACS analysis of Fcγ receptors in intact versus permeabilized mast cells showed expression of FcγRII to be 42.2 ± 3.9% and this was unchanged by permeabilization. FcγRIII protein expression was minimal and this was also unchanged by permeabilization. Aggregation of FcγRII on human mast cells led to no significant degranulation as evidenced by histamine release. Conclusions: In addition to FcγRI expression, human mast cells express FcγRIIA, FcγRIIb1, FcγRIIb2 and FcγRIII mRNA, and significant surface expression of FcγRII. Aggregation of FcγRII on cultured human mast cells in this model was not followed by histamine release.

Corticosteroids, IgE, and atopy.

Corticosteroids, IgE, and atopy.

Differential MMP and TIMP production by human marrow and peripheral blood CD34(+) cells in response to chemokines.

As stromal cell-derived factor-1 (SDF-1), macrophage inflammatory protein-1alpha (MIP-1alpha), and interleukin-8 (IL-8) are implicated in the homing and mobilization of human hematopoietic progenitors (HPC), we hypothesized that these chemokines mediate the migration of HPC across subendothelial basement membranes by regulating production of matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs). Assays for migration across reconstituted basement membrane (Matrigel) and chemotaxis were carried out using CD34(+) cells derived from normal human bone marrow (BM) and mobilized peripheral blood (PB). Secretion of MMPs and TIMPs was evaluated by zymography and reverse zymography and gene expression by RT-PCR. We found that an SDF-1 gradient increases the chemotaxis of BM and PB CD34(+) cells across Matrigel (BM > PB), which is blocked by inhibitors of MMPs (o-phenanthroline, rhTIMP-1, rhTIMP-2, and anti-MMP-9 and anti-MMP-2 antibodies) but enhanced by tumor necrosis factor-alpha (TNF-alpha), a strong stimulator of MMPs. Preincubation of these cells with SDF-1 stimulated the secretion of MMP-2 and MMP-9 in BM and PB CD34(+) cells but of TIMP-1 and TIMP-2 only in PB CD34(+) cells. Preincubation with MIP-1alpha and IL-8 also stimulated the secretion of MMP-9 and MMP-2 (BM > PB), but with respect to TIMPs, the effect was reversed (PB > BM), resulting in trans-Matrigel migration of BM but not of PB CD34(+) cells. We therefore propose that MMPs and TIMPs are involved in 1) SDF-1-induced chemotaxis of human HPC across subendothelial basement membranes, and 2) MIP-1alpha- and IL-8-stimulated migration of HPC.

P-glycoprotein (encoded by multidrug resistance genes) is not required for interleukin-2 secretion in mice and humans.

P-glycoprotein (encoded by multidrug resistance genes), a member of the ATP-binding cassette transporter protein superfamily, has been shown to play a role in the secretion of cytokines. This conclusion was based upon the inhibition of cytokine secretion by anti-P-gp monoclonal antibodies. In this study, we show that anti-CD3-stimulated lymphocytes from wild-type, mdr1a knock out and mdr1ab double knock out mice produce similar amounts of IL-2, IFN-gamma, IL-4, and IL-10. In addition, Jurkat T cells that lack P-gp and MDR1-transfected Jurkat T cells (JurkatP-gp) as well as purified human peripheral blood CD4+ P-gp+ and CD4+ P-gp- and CD8+ P-gp+ and CD8+ P-gp- T cell subsets produced comparable amounts of IL-2. These data show that P-gp is not required for secretion of IL-2, IFN-gamma, IL-4, and IL-10 secretion in mice and IL-2 secretion in humans.