Abha Saini

Age-related impairment of early and late events of signal transduction in mouse immune cells

The effects of aging were studied on both early and late [subsequent to activation of protein kinase C (PKC)] events of signal transduction in mouse T cell subsets. Aged C57Bl/6 mice showed significant suppression of Con A-stimulated Ca2+ mobilization in both CD4+ and CD8+T cells compared to young mice. When early signaling was bypassed by stimulating cells with the combination of a calcium ionophore and a phorbol ester. [3H]thymidine incorporation was comparable between young and aged mice while Con A-stimulated [3H]thymidine uptake remained depressed in aged mice. These results suggest that impairment of early (but not late) events may be responsible for the suppression of T cell proliferation in aged animals. Further, aged mice exhibited a significant increase in spontaneous proliferation resulting in a robust reduction of the stimulation index of both Con A and PMA/A23187-induced proliferation. Similarly, aged mice exhibited a significant increase in spontaneous IL-2R expression in both CD4+ and CD8+ T cells. No differences in IL-2 secretion were found between young and aged mice. The change in IL-2R expression was positively correlated with increased spontaneous proliferation. Overall these results suggest that: 1) impaired early (pre-PKC) rather than later (post-PKC) events of signaling may be responsible for poor proliferation of T cells, and 2) endogenous activation of cells related to increased level of IL-2R expression may play an important role in aging-associated immunosuppression.

Susceptibility or resistance to Leishmania infection is dictated by the macrophages evolved under the influence of IL‐3 or GM‐CSF

Although enhanced monocytopoiesis is a hallmark of leishmaniasis, its significance in determining the course of the disease has not been addressed. While the number of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐secreting cells increases in the draining lymph nodes in a resistant mouse strain (C57BL/6) during disease, in a susceptible strain (BALB/c) the number of interleukin‐3 (IL‐3)‐secreting cells increases. Treatment of BALB/c mice with anti‐IL‐3 antibody significantly reduces the disease score. Bone marrow macrophages derived under stimulation with IL‐3 (IL‐3‐Mϕ) or GM‐CSF (GM‐Mϕ) differ functionally. GM‐Mϕ are significantly more responsive to IFN‐γ‐induced augmentation and more refractory to IL‐4‐mediated suppression of anti‐leishmanial activity than IL‐3‐Mϕ. LPS‐induced IL‐12 and TNF‐α secretion by both the susceptible and resistant strain‐derived macrophage subsets are down‐regulated. Despite down‐regulation of IL‐12 secretion, GM‐Mϕ favor expansion of IFN‐γ‐secreting cells and IL‐3‐Mϕ favor IL‐6‐dependent expansion of the IL‐4‐secreting Th subset. Adoptive transfer of leishmanial antigen‐pulsed IL‐3‐Mϕ and GM‐Mϕ prior to infection either aggravated or reduced the disease score, respectively, in BALB/c mice. Anti‐IL‐6 treatment reverted the Th subset profile not only in vitro but also in vivo, resulting in a reduced disease score in both infected BALB/c mice and IL‐3‐Mϕ recipients. The disease score in IL‐3‐Mϕ recipients is also reduced significantly after anti‐IL‐4 treatment.

Preincubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34(+)CD38(-) progenitor cells.

Retroviral gene transfer studies targeting bone marrow CD34(+)CD38(-) stem cells have been disappointing because of the rarity of these cells, their G(0) cell cycle status, and their low or absent expression of surface retroviral receptors. In this study, we examined whether preincubation of bone marrow CD34(+)CD38(-) stem cells with a hematopoietically supportive porcine microvascular endothelial cell line (PMVECs) could impact the cell cycle status and expression of retroviral receptors in pluripotent CD34+CD38- cells and the efficiency of gene transfer into these primitive target cells. PMVEC coculture supplemented with GM-CSF + IL-3 + IL-6 + SCF + Flt-3 ligand induced >93% of the CD34(+)CD38(-) population to enter the G(1) or G(2)/S/M phase while increasing this population from 1.4% on day 0 to 6.5% of the total population by day 5. Liquid cultures supplemented with the identical cytokines induced 73% of the CD34(+)CD38(-) population into cell cycle but did not maintain cells with the CD34(+)CD38(-) phenotype over time. We found no significant increase in the levels of AmphoR or GaLVR mRNA in PMVEC-expanded CD34(+)CD38(-) cells after coculture. Despite this, the efficiency of gene transfer using either amphotropic vector (PA317) or GaLV vector (PG13) was significantly greater in PMVEC-expanded CD34(+)CD38(-) cells (11.4 +/- 5.6 and 10.9 +/- 5.2%, respectively) than in either steady state bone marrow CD34(+)CD38(-) cells (0.6 +/- 1.7 and 0.2 +/- 0.6%, respectively; p < 0.01 and p < 0.01) or liquid culture-expanded CD34(+)CD38(-) cells (1.4 +/- 3.5 and 0.0%, respectively; p < 0.01 and p < 0.01). Since PMVEC coculture induces a high level of cell cycling in human bone marrow CD34(+)CD38(-) cells and expands hematopoietic cells capable of in vivo repopulation, this system offers potential advantages for application in clinical gene therapy protocols.

Morphine inhibits signal transduction subsequent to activation of protein kinase C in mouse splenocytes

Morphine administered as a subcutaneous implant was previously reported to inhibit the mitogen-induced initial increases in cytoplasmic free calcium concentrations ([Ca2+]i) in mouse splenocytes. The present studies were initiated to determine whether morphine affects signal transduction subsequent to activation of protein kinase C (PKC) in immune cells. Administration of morphine significantly inhibited the phorbol myristate acetate (PMA)-stimulated increase in interleukin-2 receptor (IL-2R) expression in both CD4+ and CD8+ mouse T cells. In contrast, morphine treatment had no effect on PMA/calcium ionophore (A23187)-induced increase in IL-2 secretion, suggesting a selective inhibition of IL-2R expression. Simultaneous administration of morphine and the opiate antagonist naltrexone blocked the effect of morphine on CD4+ cells. The inhibition of PMA-stimulated IL-2R expression was not reproduced by incubating splenocytes with morphine (10(-8)-10(-5) M). These results suggest that this effect of morphine was mediated through opiate-receptors, but not directly via opiate receptors located on T cells. Moreover, adrenalectomy abolished this effect of morphine in CD4+ but not CD8+ T cells, suggesting that the inhibitory effect of morphine on IL-2R expression in CD4+ T cells may be mediated through a morphine-induced increase in corticosteroid levels. Thus, opiate-induced immunosuppression may involve an inhibition of post-PKC events, especially IL-2R expression, as well as impairment of earlier events in the activation of immune cells such as the increase in [Ca2+]i.

Hematopoietic cytokine production by human brain endothelial cells (Hubec) Is associated with the ex vivo expansion of CD34+CD38− cells

Abstract We have recently demonstrated that primary human brain endothelial cells (HUBEC) supplemented with GMCSF+IL-3+ IL-6+SCF+Flt-3 ligand support a 400-fold increase in CD34 + CD38 − cells in 7 day cultures. In order to identify soluble factors which might contribute to this hematopoietic effect, we performed ELISAs on supernatant collected from HUBEC monolayers and compared this with supernatant from human umbilical vein endothelial cells (HUVEC) which do not support the expansion of CD34 + CD38 − cells. In unconcentrated HUBEC supernatant, we detected high levels of VEGF (1951 pg/mL±561), IL-11 (1832 pg/mL±142), IL-6 (2625 pg/mL±39), and SCF (172 pg/mL±2), and surprisingly low or nonmeasurable levels of GMCSF (nm), GCSF (22 pg/mL±1), Epo (nm), IL-3 (nm), and Flt-3 ligand (18 pg/mL±0.1). In contrast, supernatant collected from HUVEC contained no measurable VEGF or IL-11, but had very high levels of GCSF (2278 pg/mL±76) and PDGF (2369 pg/mL±235). In addition, HUVEC supernatant contained IL-6 (767 pg/mL±21), GMCSF (67 pg/mL±3), and SCF (340 pg/ml±6), but Flt-3 ligand and Epo were not detectable. In summary, HUBEC secrete high levels of IL-11 and VEGF which have both been identified to induce the proliferation of hematopoietic stem cells (HSC) in vitro. In contrast, HUVEC produce neither IL-11 nor VEGF but do secrete high levels of GCSF which likely contributes to the differentiation of progenitor cells in vitro. We are currently studying the capacity of HUBEC supernatant to induce HSC proliferation in the absence of cell-to-cell contact and the contribution of known or novel cytokines toward this effect.