Homing, cell cycle kinetics and fate of transplanted hematopoietic stem cells.

Homing of transplanted hematopoietic stem cells to recipient bone marrow is a critical step in engraftment and initiation of marrow reconstitution. At present, only partial understanding of the cellular and molecular mechanisms governing homing exists. Likewise, only an incomplete list of adhesion molecules implicated in directing the trafficking of stem cells to the marrow microenvironment is available. Opposing hypotheses that attribute homing to an orderly and orchestrated cascade of events or to random migration of circulating cells find ample experimental support. Also unsettled is the fate of marrow-homed cells shortly after transplantation and the rapidity at which they begin to proliferate in their new marrow microenvironment. The limited number of studies in this field and disparities in their experimental design intensifies the confusion surrounding these critical aspects of stem cell biology. However, this area of research is moving forward rapidly and results capable of clarifying many of these issues are forthcoming.

Cis-polyunsaturated fatty acids stimulate beta1 integrin-mediated adhesion of human breast carcinoma cells to type IV collagen by activating protein kinases C-epsilon and -mu.

We have investigated the effects of various fatty acids (FAs) on integrin-mediated MDA-MB-435 breast carcinoma cell adhesion to type IV collagen (collagen IV) in vitro. Arachidonic acid (AA) and linoleic acid both induced a dose-dependent increase in cell adhesion to collagen IV with no significant increase in nonspecific adhesion to polylysine and BSA. Oleic acid (a monounsaturated FA), AA methyl ester, and linoelaidic acid (a trans-isomer of linoleic acid) failed to stimulate adhesion to collagen IV, suggesting that these effects required cis-polyunsaturation and a free carboxylic moiety and that they were not due to membrane perturbations. Calphostin C, a protein kinase C (PKC) inhibitor, blocked cis-polyunsaturated FA (cis-PUFA)-induced cell adhesion in a dose-dependent manner, suggesting a role for a calcium-dependent PKC in this signal transduction pathway. Immunoblotting revealed that cis-PUFAs induced the translocation of PKCepsilon and PKCmu, two of the novel PKC isozymes, from the cytosol to the membrane. In contrast, a conventional PKC isozyme, PKCalpha, as well as the atypical isozymes, PKCzeta and PKCiota, did not translocate after cis-PUFA treatment. Function-blocking antibodies specific for alpha1, alpha2, and beta1, integrin subunits inhibited cell adhesion to collagen IV, whereas antibodies to alpha3 and alpha5 did not. No increase in the expression of these integrins on the cell surface was detected after the incubation of cells with cis-PUFAs, suggesting that there is an increase in the activity, but not in the amount, of these beta1, integrins. Altogether, these data suggest that cis-PUFAs enhance human breast cancer cell adhesion to collagen IV by selectively activating specific PKC isozymes, which leads to the activation of beta1 integrins.

Lymphocyte-endothelial cell adhesive interactions in lung immunity: lessons from the murine response to particulate antigen.

The adhesive interaction between lymphocytes and lung endothelial cells presents an attractive arena for the development of novel therapeutic agents to modify pathologic pulmonary immune responses. The conceptual basis for choosing molecular targets to modulate this adhesive interaction derives, in large part, from results of murine experimental model systems of the pulmonary immune response. This article reviews one such model, the response of primed C57BL/6 mice to the particulate antigen sheep erythrocytes. Novel data are presented on the effect of a blocking anti-alpha(4) integrin monoclonal antibody on lung leukocyte and lymphocyte subset accumulation after intratracheal (IT) antigen challenge. Results from this model system have indicated that lymphocytes may use either the endothelial selectins or alpha(4) integrin as independent pathways to initiate recruitment into the lungs.

Alpha(1,3)-fucosyltransferase VII-dependent synthesis of P- and E-selectin ligands on cultured T lymphoblasts.

T lymphocytes up-regulate the synthesis of ligands for E- and P-selectin during proliferative responses in vivo and in vitro. Previous studies from our laboratories indicated that the alpha(1,3)-fucosyltransferase FucT-VII regulates the synthesis of E-selectin ligands and sialylated Lewis(x)-related epitopes (sLe(x)-related epitopes) in human T lymphoblasts. The current report shows that production of both P- and E-selectin ligands is FucT-VII dependent, but peak synthesis of each occurs at different levels of fucosyltransferase activity in intact cells. In brief, FucT-VII mRNA levels were higher in cultured T lymphoblasts expressing sLe(x)-related epitopes and both selectin ligands than in cells expressing P-selectin ligands alone. However, synthesis of the epitopes and both selectin ligands required the FucT-VII enzyme in transfected Molt-4 cells. In contrast, neither constitutive nor transfection-enhanced levels of the FucT-IV enzyme generated active P-selectin ligands in these lines. In addition, targeted deletion of the FucT-VII gene in mice markedly inhibited the synthesis of both P- and E-selectin ligands during blast transformation in vitro. Finally, the optimal synthesis of active P-selectin ligands occurred at lower level of FucT-VII activity than required for synthesis of equally active E-selectin ligands in both cultured T lymphoblasts and FucT-VII transfectants. Consequently, the FucT-VII enzyme is essential for the synthesis of both P- and E-selectin ligands by T lymphoblasts, and its activity determines whether P-selectin ligands are expressed alone or in conjunction with E-selectin ligands and sLe(x)-related epitopes on human T cells.

Endothelial selectins and alpha4 integrins regulate independent pathways of T lymphocyte recruitment in the pulmonary immune response.

The cell adhesion molecules (CAMs) required for T lymphocyte recruitment during pulmonary immune responses have not been defined. Our laboratories recently reported that intratracheal (IT) challenge of sensitized mice with SRBC induced prolonged expression of vascular P-selectin, E-selectin, and VCAM-1, particularly in areas of mononuclear leukocyte infiltration. A surge in the number of circulating T lymphocytes expressing selectin ligands preceded the peak accumulation of T cells in the lung. In addition, a significant percentage of the T cells recovered from the lung expressed selectin ligands as well. The current study demonstrates that cultured T lymphoblasts use both selectin ligands and alpha4 integrins to enter the airspace and interstitium during the response to SRBC. Fluorescently labeled T lymphoblasts, derived via activation on CD3 and growth in low dose IL-2, showed inflammation-specific recruitment into lungs harvested 24 h after cell infusion. Their flux paralleled the accumulation of host lymphocytes in the lung, with both peaking 2 to 4 days after SRBC challenge. Trafficking studies conducted over a 24-h period during peak lymphocyte accumulation in the lungs revealed preferential recruitment of labeled T lymphoblasts expressing P- and E-selectin ligands. In addition, mAb blockade of the alpha4 integrins and targeted deletion of an alpha(1,3)fucosyltransferase essential for selectin ligand synthesis each reduced labeled T lymphoblast trafficking to a significant degree. Furthermore, alpha4 integrin blockade reduced the trafficking of the selectin ligand-deficient cells into the airspace, confirming that its contribution is in part independent from the vascular selectins. These findings imply that both selectin ligands and alpha4 integrins participate in T lymphoblast recruitment during the pulmonary immune response to IT SRBC.

Lymphocyte recruitment and the kinetics of adhesion receptor expression during the pulmonary immune response to particulate antigen.

The selectins and beta2 integrins participate in the recruitment of neutrophils in acute pulmonary inflammation. However, the cell adhesion receptors that mediate lymphocyte trafficking into the lung have not been defined. This study examined the relationship between cell adhesion molecules on the pulmonary vasculature and on lymphocytes recovered from the lung during a pulmonary immune response to intratracheal (I.T.) sheep red blood cells (SRBCs) in sensitized C57BL/6J mice. Silver-enhanced immunogold staining and reverse transcriptase polymerase chain reaction of lung tissues revealed sustained induction of VCAM-1, E-selectin, and P-selectin on the pulmonary vasculature for up to 7 days after I.T.-SRBC challenge. Neither the MECA 79 nor MECA 367 antigens were induced on the pulmonary vasculature during this period. In the peripheral blood, both CD4 and CD8 T-cell subsets showed an initial increase in P-selectin ligand expression after I.T.-SRBC challenge. The number of P-selectin ligand-positive T cells in the peripheral blood fell as T cells with both P-selectin and, to a lesser extent, E-selectin ligands accumulated in the bronchoalveolar lavage fluid. We conclude that I.T.-SRBC challenge in sensitized mice elicits prolonged synthesis of P-selectin, E-selectin, and VCAM-1 by the lung vasculature as well as selectin ligand synthesis by responding T cells. Furthermore, the entry of selectin-ligand-positive T cells into the circulation and their accumulation in the bronchoalveolar lavage fluid indicates that these receptors may contribute to T cell recruitment. Finally, VCAM-1 on the vasculature may also participate; however, the vascular addressins, required for homing to peripheral and mucosal lymphoid organs, are not essential for T-cell entry into the lung following I.T.-SRBC challenge.

A simple method for the purification of human peripheral blood monocytes. A substitute for Sepracell-MN.

Sepracell-MN has provided a simple, rapid means of isolating peripheral blood monocytes. However this product is no longer available. Consequentially we have developed a Percoll gradient which matches Sepracell-MN in simplicity and yield of monocytes. Using this Percoll gradient, an average of 7 x 10(6) monocytes with a purity of 83% were obtained from 30-40 ml of blood. These monocytes were at least 97% viable and responded to chemotactic stimuli in comparable numbers to those prepared using Sepracell-MN.

Secretion of monocyte chemotactic activity by alveolar macrophages.

The purpose of this study was to determine if alveolar macrophages (AMs) are a source of monocyte chemoattractants and the role bleomycin interaction with AMs may play in the recruitment of monocytes to the lung in a rodent model of bleomycin-induced pulmonary fibrosis. AMs isolated from rats with bleomycin-induced fibrosis secreted significantly greater amounts of monocyte chemoattractants than those isolated from normal rats. When AMs from normal rats were stimulated with bleomycin in vitro, monocyte chemotactic activity was secreted into the medium. Chemotactic activity secretion by AM stimulated with 0.01 to 0.1 micrograms/ml bleomycin was significantly higher than that of cells incubated in medium alone. This activity was truly chemotactic for monocytes, but caused only minimal migration of normal AMs. Bleomycin itself at concentrations of 1 pg/ml to 10 micrograms/ml had no monocyte chemoattractant activity. Characterization of the chemotactic activity in conditioned media (CM) from bleomycin-stimulated AM demonstrated that the major portion of the activity bound to gelatin, was heterogeneous, with estimated molecular weights of 20 to 60 kd, and was inactivated by specific antifibronectin antibody. These findings suggest that fibronectin fragments are primarily responsible for the monocyte chemotactic activity secreted by AMs. Through increased secretion of such chemotactic substances, AMs could play a key role in the recruitment of peripheral blood monocytes into the lung in inflammatory lung disease and fibrosis.