Changes in cell migration-related molecules expressed by thymic microenvironment during experimental Plasmodium berghei infection: consequences on thymocyte development.

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

[Effect of epidermal growth factor signal pathway on integrin alpha5 beta1 in prostate cancer cell line DU145].

OBJECTIVE: To investigate the molecular mechanism of epidermal growth factor (EGF) signal pathway on the expression of integrin alpha5 beta1 in prostate cancer cell line DU145. METHODS: Using flow-cytometry, the effects of EGF and the mitogen-activated protein kinase (MAPK) signal pathway inhibitor PD98059 on the expression of integrin alpha5 and beta1 subunits on DU145 cell surface were analyzed. RT-PCR and Western blot methods were used to examined the expression of mRNA and cell total protein of integrin alpha5 and beta1 subunits. And the metastatic phenotypes in DU145 cell were investigated. RESULTS: The expression levels of integrin alpha5 beta1, which was the receptor for fibronectin, were changed. EGF up-regulated the protein and mRNA expression of beta1 subunit on DU145 cell surface, 231% and 248% (P < 0.01) compared to the control respectively, and it could significantly promote the ability of DU145 cell adhesion to fibronectin and migration. However PD98058, which was the inhibitor of MAPK signal pathway, down-regulated the protein and mRNA expression of beta1 subunit, 60% and 63% (P < 0.01) compared to the control respectively, and it had the contrary function on the adhesion and migration ability of DU145 cell. But both had no effect the expression of alpha5 subunit. CONCLUSIONS: EGF might promote the metastatic ability mainly by up-regulating the expression of beta1 subunit by activating MAPK signal pathway in DU145 cells. Their regulation effects are on the mRNA transcriptional level.

Characterization of integrin and anchorage dependence in mammary epithelial cells following c-erbB2-induced epithelial-mesenchymal transition.

Signalling from the proto-oncogene c-erbB2 in mammary epithelial cells has earlier been shown to result in epithelial-mesenchymal transition (EMT) giving rise to fibroblast-like cells, and acquisition of anchorage-independent growth (AIG) usually determined by growth capacity in soft agar. In this study, we have analysed AIG associated with c-erbB2-induced EMT in a human mammary epithelial cell line. Intriguingly, cells capable of growth in soft agar were shown to be dependent on the function of beta(1) integrin extracellular matrix receptors for growth in collagen. We therefore tested the hypothesis that apparent AIG was due to deposition of extracellular matrix in the agar. Although the fibroblastic cells had strongly upregulated expression of the fibronectin receptor subunit integrin alpha(5) andabundant fibronectin fibrils, these properties did not have a positive correlation with AIG. Furthermore, antibody blocking of integrin alpha(5) and beta(1) failed to inhibit AIG. These results indicate that the anchorage-independent cells are not dependent on connection to extracellular matrix, but instead may be subject to a growth-inhibitory effect from the collagen in the absence of integrin signalling. This notion was supported by the finding that integrin blocking of the fibroblastic cells in fibrin was without effect on proliferation.

Impaired migration of NOD mouse thymocytes: a fibronectin receptor-related defect.

We previously showed intrathymic alterations in non-obese diabetic (NOD) mice, including the appearance of giant perivascular spaces, filled with mature thymocytes, intermingled with an extracellular matrix network. This raised the hypothesis of a defect in thymocyte migration with partial arrest of exiting thymocytes in the perivascular spaces. Herein, we investigated the expression of receptors for fibronectin [very late antigen (VLA)-4 and VLA-5] and laminin (VLA-6), known to play a role in thymocyte migration. When compared with two normal and one other autoimmune mouse strains, a decrease of VLA-5 expression in NOD thymocytes was noticed, being firstly observed in late CD4/CD8 double-negative cells, and more pronounced in mature CD4(+) and CD8(+) thymocytes. Functionally, thymocyte exit from the lymphoepithelial complexes, the thymic nurse cells, was reduced. Moreover, NOD thymocyte adhesion to thymic epithelial cells as well as to fibronectin was diminished, and so was the migration of NOD thymocytes through fibronectin-containing transwell chambers. In situ, intra-perivascular space thymocytes were VLA-5-negative, suggesting a correlation between the thymocyte arrest within these structures and loss of VLA-5 expression. Overall, our data reveal impairment in NOD thymocyte migration, and correspond to the first demonstration of a functional fibronectin receptor defect in the immune system.

Sensory neuron subtypes have unique substratum preference and receptor expression before target innervation.

The factors controlling the specification and subsequent differentiation of sensory neurons are poorly understood. Data from embryological manipulations suggest that either sensory neuron fates are specified by the targets they encounter or sensory neurons are considerably more "plastic" with respect to specification than are neurons of the CNS. The prevailing view that sensory neurons are specified late in development is not consistent, however, with the directed outgrowth of sensory neurons to their targets and the characteristic spatial distribution of sensory neuron fates within the peripheral ganglia. To address when in development different classes of sensory neurons can first be distinguished, we investigated the interactions of early dorsal root ganglia neurons with the extracellular matrix before neurite outgrowth to targets. We found that subclasses of sensory neurons in early dorsal root ganglia show different patterns of neurite outgrowth and integrin expression that are predictive of their fates. In the absence of neurotrophins, presumptive proprioceptive neurons extend neurites robustly on both laminin and fibronectin, whereas presumptive cutaneous neurons show a strong preference for laminin. Cutaneous afferents that have innervated targets show a similar strong preference for laminin and show higher levels of integrin alpha7beta1 than do proprioceptive neurons. Finally, presumptive proprioceptive neurons express fibronectin receptors, integrin alpha3beta1, alpha4beta1, and alpha5beta1, at higher levels than do presumptive cutaneous neurons. Our results indicate that subtypes of sensory neurons have unique patterns of neurite outgrowth and receptor expression before target innervation.

Central roles of alpha5beta1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies.

Vascular development and maturation are dependent on the interactions of endothelial cell integrins with surrounding extracellular matrix. Previous investigations of the primacy of certain integrins in vascular development have not addressed whether this could also be a secondary effect due to poor embryonic nutrition. Here, we show that the alpha5 integrin subunit and fibronectin have critical roles in blood vessel development in mouse embryos and in embryoid bodies (EBs) differentiated from embryonic stem cells (a situation in which there is no nutritional deficit caused by the mutations). In contrast, vascular development in vivo and in vitro is not strongly dependent on alpha(v) or beta3 integrin subunits. In mouse embryos lacking alpha5 integrin, greatly distended blood vessels are seen in the vitelline yolk sac and in the embryo itself. Additionally, overall blood vessel pattern complexity is reduced in alpha5-null tissues. This defective vascular phenotype is correlated with a decrease in the ligand for alpha5 integrin, fibronectin (FN), in the endothelial basement membranes. A striking and significant reduction in early capillary plexus formation and maturation was apparent in EBs formed from embryonic stem cells lacking alpha5 integrin or FN compared with wild-type EBs or EBs lacking alpha(v) or beta3 integrin subunits. Vessel phenotype could be partially restored to FN-null EBs by the addition of whole FN to the culture system. These findings confirm a clear role for alpha5 and FN in early blood vessel development not dependent on embryo nutrition or alpha(v) or beta3 integrin subunits. Thus, successful early vasculogenesis and angiogenesis require alpha5-FN interactions.

The alpha5beta1 integrin selectively enhances epidermal growth factor signaling to the phosphatidylinositol-3-kinase/Akt pathway in intestinal epithelial cells.

We have investigated EGF-driven signaling processes in rat intestinal epithelial cell lines that overexpress either the alpha5beta1 integrin or the alpha2beta1 integrin. Both cell types display efficient activation of Erk/MAP kinase, but only the alpha5beta1 expressing cells display a strong activation of Akt. A complex is formed between activated EGFR and alpha5beta1, but not with alpha2beta1; this complex also contains ErbB3 and p85. Thus alpha5beta1 can support efficient activation of both the Erk and the phosphatidylinositol-3-kinase/Akt branches of the EGFR signaling cascade, whereas alpha2beta1 can support only the Erk branch.

Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells.

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10-induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

Interference of tenascin-C with syndecan-4 binding to fibronectin blocks cell adhesion and stimulates tumor cell proliferation.

Tenascin-C is an adhesion-modulatory extracellular matrix molecule that is highly expressed in tumors. To investigate the effect of tenascin-C on tumor cells, we analyzed its antiadhesive nature and effect on tumor cell proliferation in a fibronectin context. Glioblastoma and breast carcinoma cell adhesion was compromised by a mixed fibronectin/tenascin-C substratum, which concomitantly caused increased tumor-cell proliferation. We identified the antiadhesive mechanism as a specific interference of tenascin-C with cell binding to the HepII/syndecan-4 site in fibronectin through direct binding of tenascin-C to the 13th fibronectin type III repeat (FNIII13). Cell adhesion and proliferation levels were restored by the addition of FNIII13. Overexpression of syndecan-4, but not syndecan-1 or -2, reverted the cell adhesion defect of tenascin-C. We characterized FNIII13 as the binding site for syndecan-4. Thus we describe a novel mechanism by which tenascin-C impairs the adhesive function of fibronectin through binding to FNIII13, thereby inhibiting the coreceptor function of syndecan-4 in fibronectin-induced integrin signaling.

Impairment of gingival fibroblast adherence by IL-6/sIL-6R.

Interleukin-6 (IL-6) binds to human gingival fibroblasts (HGF) in the presence of a soluble form of IL-6 receptor (sIL-6R). We investigated the effects of IL-6 on the functions of HGF in the presence of sIL-6R. HGF changed their morphology from spindle-shaped to round, and detached from the culture dish by stimulation with IL-6/sIL-6R. In this condition, a signal transducer gp130 and a transcription factor Stat3 were phosphorylated, resulting in activation of transcription factors Stat3 and C/EBPbeta. Cytoskeletal beta-actin and adhesion molecule integrin-alpha5, a subunit of alpha5beta1 integrin (VLA-5), were found to possess potential binding domains for these transcription factors in their promoters. Accumulation of beta-actin and integrin-alpha5 mRNA decreased, contrary to the expectation of the induction of gene transcription. Furthermore, the decrease in their mRNAs was associated with reduced expression of both actin and VLA-5 proteins. These results suggest that the expression of VLA-5 and actin was down-regulated in HGF through an IL-6 signaling pathway, resulting in impairment of HGF adherence.

Integrin expression, enterocyte maturation, and bacterial internalization.

BACKGROUND: Little is known about the molecular mechanisms involved in the translocation of enteric bacteria. Adhesion molecules mediate interactions between some enteric pathogens and mammalian cells, but no such interactions have been identified for enterocytes and normal enteric bacteria. Using enteric pathogens, adhesion molecule expression has been linked to bacterial internalization and to enterocyte differentiation. Therefore, experiments were designed to study enterocyte integrin expression and differentiation, as well as enterocyte internalization of Salmonella typhimurium, Proteus mirabilis, and Escherichia coli. MATERIALS AND METHODS: Relative expression of the alpha2, alpha3, and beta1 integrin subunits on Caco-2 and HT-29 enterocytes (mature and immature) was measured by ELISA. Bacteria-enterocyte surface interactions were observed by light and scanning electron microscopy. Bacterial internalization by enterocytes was quantified using the gentamicin protection assay. RESULTS: Expression of the alpha2, alpha3, and beta1 integrin subunits was consistently increased in immature compared to mature Caco-2 enterocytes; however, compared to mature enterocytes, immature HT-29 enterocytes had similar expression of alpha3 and beta1 but decreased alpha2. Compared to untreated mature enterocytes, bacterial internalization was increased in immature enterocytes as well as mature enterocytes with lateral membranes artifactually exposed. However, there was no difference in bacterial internalization between immature enterocytes and mature enterocytes treated to expose the lateral membrane. CONCLUSIONS: Bacterial internalization by enterocytes appeared to be due to factors other than integrin expression or enterocyte differentiation. Exposure of the lateral enterocyte membrane may play an important role in facilitating bacterial internalization by enterocytes.

Adult neuronal regeneration induced by transgenic integrin expression.

In a variety of adult CNS injury models, embryonic neurons exhibit superior regenerative performance when compared with adult neurons. It is unknown how young neurons extend axons in the injured adult brain, in which adult neurons fail to regenerate. This study shows that cultured adult neurons do not adapt to conditions that are characteristic of the injured adult CNS: low levels of growth-promoting molecules and the presence of inhibitory proteoglycans. In contrast, young neurons readily adapt to these same conditions, and adaptation is accompanied by an increase in the expression of receptors for growth-promoting molecules (receptors of the integrin family). Surprisingly, the regenerative performance of adult neurons can be restored to that of young neurons by gene transfer-mediated expression of a single alpha-integrin.

Tumour necrosis factor-alpha provokes upregulation of alpha2beta1 and alpha5beta1 integrins, and cell migration in OST osteosarcoma cells.

OST cells enhance the induction of matrix metalloproteinase (MMP)-9 by tumour necrosis factor (TNF)-alpha and the corresponding metastasis to lungs in vivo (Kawashima et al., 1994). We focused on the adhesive and migratory properties of OST cells, and investigated the expression of integrins in OST cells stimulated by TNFalpha in vitro. OST cells potentiated not only adhesion to the extracellular matrix (ECM) but also the migration on ECM. On competitive reverse transcription-polymerase chain reaction (RT-PCR) analyses, the amounts of alpha2 (4.9-fold), alpha5 (1.2-fold) and alpha(v) (4.9-fold) were upregulated by TNFalpha at the transcriptional level. Alpha-5 showed a slight increase by flow cytometry; however, alpha2 and alphav integrins remained unchanged at the protein level. Immunofluorescence study disclosed integrins of alpha2beta1 and alpha5beta1 were much clustered at cell processes by TNFalpha stimulation, probably related to increased cell adhesion and migration. Therefore, the upregulation of alpha2beta1 and alpha5beta1 integrins seems to contribute to tumour invasion and metastatic potential.

Keratinocyte growth factor induces hyperproliferation and delays differentiation in a skin equivalent model system.

Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell growth. To examine the direct effects of KGF on the morphogenesis of the epidermis, we generated skin equivalents in vitro by seeding human keratinocytes on the papillary surface of acellular dermis and raising them up to the air-liquid interface. KGF was either added exogenously or expressed by keratinocytes via a recombinant retrovirus encoding KGF. KGF induced dramatic changes to the 3-dimensional organization of the epidermis including pronounced hyperthickening, crowding, and elongation of the basal cells, flattening of the rete ridges, and a ripple-like pattern in the junction of stratum corneum and granular layers. Quantitative immunostaining for the proliferation antigen, Ki67, revealed that in addition to increasing basal proliferation, KGF extended the proliferative compartment by inducing suprabasal cell proliferation. KGF also induced expression of the integrin alpha 5 beta 1 and delayed expression of keratin 10 and transglutaminase. However, barrier formation of the epidermis was not disrupted. These results demonstrate for the first time that a single growth factor can alter the 3-dimensional organization and proliferative function of an in vitro epidermis. In addition to new strategies for tissue engineering, such a well-defined system will be useful for analyzing growth factor effects on the complex links between cell proliferation, cell movement and differentiation within a stratified tissue.

Cardiac expression of a gain-of-function alpha(5)-integrin results in perinatal lethality.

Communication between the extracellular matrix and the intracellular signal transduction and cytoskeletal system is mediated by integrin receptors. alpha(5)beta(1)-Integrin and its cognate ligand fibronectin are essential in development of mesodermal structures, myocyte differentiation, and normal cardiac development. To begin to explore the potential roles of alpha(5)beta(1)-integrin specifically in cardiomyocytes, we used a transgenic expression strategy. We overexpressed two forms of the human alpha(5)-integrin in cardiomyocytes: the full-length wild-type alpha(5)-integrin and a putative gain-of-function mutation created by truncating the cytoplasmic domain, designated alpha(5-1)-integrin. Overexpression of the wild-type alpha(5)-integrin has no detectable adverse effects in the mouse, whereas expression of alpha(5-1)-integrin caused electrocardiographic abnormalities, fibrotic changes in the ventricle, and perinatal lethality. Thus physiological regulation of integrin function appears essential for maintenance of normal cardiomyocyte structure and function. This strengthens the role of inside-out signaling in regulation of integrins in vivo and suggests that integrins and associated signaling molecules are important in cardiomyocyte function.

[Quantitative expression of the adhesion receptors VLA-4, VLA-5, L-selectin, MAC-1, and ICAM-1 on the surface of CD34+ cells]. / Expression quantitative des récepteurs d'adhérence VLA-4, VLA-5, L-sélectine, MAC-1, et ICAM-1 à la surface des cellules CD34+.

The aim of this work was to quantify by flow cytometry the main adhesion receptors on CD34+ cells. These cells were isolated from bone marrow (BM) or mobilized peripheral blood (PB). The proportions of CD34+/CD49d+ and CD34+/CD49e+ are weaker on PB cells, without quantitative expression variation. This phenotypic variation may induce CD34+ cells exist from BM into circulation, promoting the mobilization. The homing to the BM implicate the CD62L receptor, which expression was found more frequently and stronger on PB cells than on BM. The CD11b, CD18 and CD54 receptors are implicated in CD34+ cells adhesion to BM micro-environment. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. Moreover, CD54 receptor was more frequently expressed on PB cells. Quantitative analysis revealed that CD18 was more strongly expressed on BM than on PB cells. This quantitative variation could promote progenitor adhesion by interacting with stromal cells. Finally, quantitative expression of the main receptors on CD34+ cells provides an original option for studying CD34+ cells during the mobilization, the homing or the adhesion to BM micro-environment.

Integrin modulating factor: a 30-kD protein that modulates the expression and function of alpha5beta1 integrin receptor.

The integrin family of cell surface receptors consists of transmembrane glycoproteins involved in cellular morphology, cytoarchitecture, cell-cell, and cell-extracellular matrix interaction. Changes in integrin receptor expression are associated with malignant transformation. The adhesion promoting activity of several members of the integrin receptors may be modulated. Integrin Associated Proteins and integrin modulating factor that may modulate integrin receptors expression and function have been reported. In this article, we report the identification of a 30-kD protein produced in SiHa cell culture medium that can modulate the expression and function of alpha5beta1 integrin receptor in HeLaS3 cells. The cell adhesion assay clearly demonstrated that HeLaS3 cells grown in a serum-free culture medium of SiHa cells (fresh medium: culture medium = 3:1) stimulated the ligand binding activity of alpha5beta1 receptor to fibronectin in a time-dependent manner, having a peak activity at 72 hours of culture. Immunocytochemical localization showed a very high expression of alpha5beta1 receptor in HeLaS3 cells grown in a SiHa culture medium for 72 hours. The (NH4)2SO4 fractionation demonstrated that proteins present in 80-100% (NH4)2SO4 saturated fraction of serum-free SiHa culture medium have a significant stimulatory effect on the binding of HeLaS3 cells to fibronectin ligand via the alpha5beta1 integrin receptor. High pressure liquid chromatography (HPLC) separation of 80-100% (NH4)2SO4 saturated fraction showed a 30- kD protein in polyacrylamide gel electrophoresis (PAGE) analysis that has a maximum stimulatory effect on the binding of HeLaS3 cells to fibronectin ligand via the alpha5beta1 integrin receptor. In conclusion, our observations indicated that human cervical tumor cells SiHa produce a 30-kD protein that can modulate the expression and function of alpha5beta1 fibronectin integrin receptor of HeLaS3 cells. These findings strengthen the concept that some cellular proteins, also called Integrin Associated Protein, may regulate the integrin receptor expression and function. Studies are in progress to characterize this 30-kD integrin modulating factor and its role in the regulation of integrin receptor function.

[The adhesive and migrating function of human epithelial cells opsonized by fibronectin].

OBJECTIVE: To investigate the adhesive and migrating function of human epithelial keratiocytes during in vitro culture. METHODS: The adhesive and migrating function was determined in freshly isolated EKCs and those cultured for 7 similar 10 days after being opsonized by fibronectin (FN). The expressions of alpha(5)beta(1) receptors in EKC were examined with indirect immunofluorescence staining methods before and after culture. RESULTS: (1) The adhesive and migrating indices after FN opsonization of EKCs freshly isolated EKCs were obviously lower than those cultured for 7 similar 10 days (P < 0.01). (2) There exhibited positive staining of the expression of alpha(5)beta(1) receptors in the EKC after 1 day culture and in the proliferative epithelia after in vitro culturing of a tissue mass. While there exhibited strongly positive staining of the peripheral proliferative epithelia of the EKCs and tissue mass after 7 days of culture, negative or weakly positive stainings were found in freshly isolated EKCs. CONCLUSION: (1) There existed significant difference of biological function between the freshly isolated EKCs and those cultured for 7 similar 10 days. (2) The strongest expression of alpha(5)beta(1) receptors was observed at the active proliferative site (peripheral proliferative epithelia of a tissue mass) and in the biological active period (cultured for 7 days) of EKCs. (3) The adhesive and migrating function of EKCs could be effectively induced and activated by in vitro culture, which enabled the EKC to alter from a relatively biologically functional static state to an actively functional state.

Ultrastructural study of expression of adhesion molecules between blood monocytes and alveolar macrophages from patients with pulmonary sarcoidosis.

Pulmonary sarcoidosis is a chronic inflammatory disorder characterized by the presence of activated T cells and alveolar macrophages at sites of inflammation. These cells are recovered from bronchoalveolar lavage (BAL) from sarcoid patients in order to evaluate the expression of various markers on cell surfaces that should determine the diagnosis in sarcoidosis. In this work we compared the expression of VLA-4, VLA-5, Mac-1, ICAM-1 and VCAM- 1 adhesion molecules, at ultrastructural level, between blood monocytes and alveolar macrophages obtained from BAL, from patients with pulmonary sarcoidosis. Cells obtained from blood and BAL were fixed, embedded in LRWhite and then ultrathin sections were incubated with monoclonal antibodies against VLA-4, VLA-5, Mac-1, ICAM-1 and VCAM-1. The results showed a more evident labelling of all adhesion molecules on alveolar macrophages when compared to blood monocytes. The labelling was seen at cell surface, at cytoplasm and small vacuoles. The differences on adhesion molecule distributions from blood monocytes to alveolar macrophages suggest that changes in the expression of these molecules occur during pulmonary inflammatory response. Lymphocytes from BAL or blood had a weak label for these molecules.

Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0).

An understanding of mechanisms regulating hematopoietic stem cell engraftment is of pivotal importance to the clinical use of cultured and genetically modified transplants. Human cord blood (CB) cells with lymphomyeloid repopulating activity in NOD/SCID mice were recently shown to undergo multiple self-renewal divisions within 6 days in serum-free cultures containing Flt3-ligand, Steel factor, interleukin 3 (IL-3), IL-6, and granulocyte colony-stimulating factor. The present study shows that, on the fifth day, the transplantable stem cell activity is restricted to the G(1) fraction, even though both colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) in the same cultures are approximately equally distributed between G(0)/G(1) and S/G(2)/M. Interestingly, the G(0) cells defined by their low levels of Hoechst 33342 and Pyronin Y staining, and reduced Ki67 and cyclin D expression (representing 21% of the cultured CB population) include some mature erythroid CFCs but very few primitive CFCs, LTC-ICs, or repopulating cells. Although these findings suggest a cell cycle-associated change in in vivo stem cell homing, the cultured G(0)/G(1) and S/G(2)/M CD34(+) CB cells exhibited no differences in levels of expression of VLA-4, VLA-5, or CXCR-4. Moreover, further incubation of these cells for 1 day in the presence of a concentration of transforming growth factor beta(1) that increased the G(0)/G(1) fraction did not enhance detection of repopulating cells. The demonstration of a cell cycle-associated mechanism that selectively silences the transplantability of proliferating human hematopoietic stem cells poses both challenges and opportunities for the future improvement of ex vivo-manipulated grafts. (Blood. 2000;96:4185-4193)