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K S Landreth

Publications and source records attributed to K S Landreth.

At least 19 recordsLinked to original sources
PubMed2000

Effects of the protein tyrosine phosphatase CD45 on FcgammaRIIa signaling and neutrophil function.

OBJECTIVE: Neutrophil receptors for the Fc portion of IgG (FcgammaR) trigger immune responses following cross-linking by IgG-coated foreign particles or immune complexes. Membrane-associated CD45, a protein tyrosine phosphatase termed leukocyte common antigen, has been shown to be essential for antigen receptor kinase mediated signaling in lymphocytes, and we hypothesized that CD45 may play a similar role in FcgammaR-mediated signaling and immune function in human neutrophils. METHODS: The experimental approach was that of cell surface molecule ligation via cross-linking with specific antibodies. Antibody dependent cellular cytotoxicity (ADCC) was assessed using a single-cell plaque assay and IL-6 production measured using ELISA. Tyrosine phosphorylation levels were assessed with anti-phospho-tyrosine blots and F-actin polymerization by flow cytometry and confocal microscopy. RESULTS: Neutrophils pretreated with anti-CD45 had a reduced ability to perform ADCC compared to untreated neutrophils. FcgammaRIIa cross-linking resulted in significantly increased concentrations of secreted IL-6 compared to untreated neutrophils, and IL-6 production was further enhanced by cocross-linking CD45 with FcgammaRIIa. Cross-linking CD45 alone also induced IL-6 production. FcgammaRIIa cross-linking resulted in increased protein tyrosine phosphorylation and F-actin polymerization in neutrophils. Cocross-linking CD45 with FcgammaRIIa resulted in abrogation of FcgammaRIIa mediated tyrosine phosphorylation and F-actin polymerization. CONCLUSIONS: These data provide evidence that CD45 can regulate or enhance the stimulation and function of human neutrophils mediated through FcgammaR(s). In addition, CD45 ligation may play an essential role in cytokine induction pathways that lead to inflammatory reactions in vivo.

Actins↗
PubMed2000

IL-5 production by bone marrow stromal cells: implications for eosinophilia associated with asthma.

BACKGROUND: Eosinophil infiltration of bronchial tissue is a hallmark of asthma. Recruitment of eosinophils into pulmonary tissue is dependent on the presence of IL-5. In addition, IL-5 plays a significant role in the differentiation, proliferation, and maturation of eosinophil progenitor cells in the bone marrow before recruitment into the lung. The contribution of bone marrow eosinophil production to eosinophilia associated with asthma is poorly understood. OBJECTIVE: The aims of this study were to determine whether bone marrow stromal cells produce IL-5 and to determine whether IL-5 production by stromal cells is upregulated by IL-1, an inflammatory cytokine associated with asthma. METHODS: IL-5 messenger (m)RNA from bone marrow stromal cells was amplified by RT-PCR and sequenced. Stromal cells were lysed, and IL-5 protein production was measured by ELISA. Upregulation of stromal cell IL-5 transcription, translation, and functional effect on eosinophil differentiation was evaluated after stimulation with recombinant IL-1alpha and IL-1beta and compared with untreated cells. RESULTS: Bone marrow stromal cells transcribe and translate IL-5. The nucleotide sequence of IL-5 mRNA from stromal cells was identical to that previously reported for murine T cells. IL-5 mRNA abundance in stromal cells increased with increasing cell confluence in culture. IL-5 mRNA and protein levels were upregulated by exposure of stromal cells to the inflammatory cytokines IL-1alpha and IL-1beta. Exposure of stromal cells to IL-1 resulted in increased eosinophil differentiation in coculture experiments with nonadherent bone marrow cells. CONCLUSION: The production of IL-5 mRNA and protein by bone marrow stromal cells is a novel finding that has implications for both normal eosinophilopoiesis and development of the accelerated eosinophil production associated with asthma.

Animals↗
PubMed1999

Regulation of BAX and BCL-2 expression in breast cancer cells by chemotherapy.

Optimizing chemotherapeutic drug delivery strategies relies, in part, on identification of the most clinically effective sequence, dose, and duration of drug exposure. The combination of dose intensive etoposide (VP-16) followed by cyclophosphamide has clinical efficacy in the treatment of advanced breast cancer. However, molecular mechanisms that underlie the effectiveness of this combination of chemotherapeutic agents have not been investigated. In this study we investigated regulation of BAX and BCL-2 expression by VP-16 and cyclophosphamide as a potential mechanism for the induction of breast cancer cell death induced by this regimen. There was a dose and time dependent increase in BAX expression in the breast cancer cell lines MCF-7, MDA-MB-435S, and MDA-MB-A231 following in vitro treatment with 50-100 microM VP-16. Elevation of BAX protein expression in the presence of VP-16 alone did not correlate with reduced viability or induction of apoptosis in MCF-7, MDA-MB-435S, or MDA-MB-A231. VP-16 did effectively block the breast cancer cell lines evaluated (MCF-7 and MDA-MB-435S) at G2/M phase of the cell cycle, confirming activity of the drug in vitro. MCF-7 and MDA-MB-435S cells that were pre-treated with VP-16 and subsequently exposed to 1.0-12.0 microg/ml 4-hydroperoxycyclophosphamide (4HC), an active metabolite of cyclophosphamide, had markedly reduced viability when compared to matched controls treated with either VP-16 or 4HC individually. Consistent with this loss of viability, exposure of all three cell lines to the combination of VP-16 and 4HC resulted in higher BAX protein levels than those observed following treatment with either single agent. This combination of chemotherapeutic agents also resulted in reduced BCL-2 expression. These observations suggest that combination chemotherapy may derive its efficacy, in part, through coordinated regulation of specific gene products associated with apoptosis. Characterization of molecular events that underlie susceptibility of specific tumor cells to combination chemotherapeutic regimens may lead to additional improvements in treatment strategies for this disease.

Adenocarcinoma↗
PubMed1998

Interleukin-6 production by human neutrophils after Fc-receptor cross-linking or exposure to granulocyte colony-stimulating factor.

Polymorphonuclear neutrophils (PMNs) are essential effector cells in host defense and tissue inflammatory responses. These responses may be initiated after cross-linking of cell surface Fc receptors that bind the constant portion of IgG (FcgammaR). We evaluated the effect of cross-linking FcgammaRI or FcgammaRII on interleukin-6 (IL-6) production by purified PMNs from normal donors or from patients being treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF). In PMNs from normal donors, IL-6 mRNA was detected by reverse transcriptase-polymerase chain reaction only after FcgammaRI or FcgammaRII cross-linking. We also found that IL-6 mRNA could be detected in PMNs after either in vitro or in vivo rhG-CSF treatment in the absence of FcgammaR cross-linking. IL-6 protein was found to be produced intracellularly and secreted by PMNs after cross-linking FcgammaRI or FcgammaRII or after rhG-CSF stimulation. Cross-linking FcgammaRI or FcgammaRII on PMNs from patients treated with rhG-CSF resulted in a synergistic increase in IL-6 secretion. Upregulation of IL-6 production by PMNs after rhG-CSF treatment may contribute to a clinical engraftment syndrome that occurs during periods of rapid increase in PMN numbers in patients receiving rhG-CSF.

Gene Expression Regulation↗
PubMed1997

Disruption of bone marrow stromal cell function by etoposide.

Long-term deficits in hematopoietic reconstitution following aggressive chemotherapeutic regimens used before transplantation may be a result, in part, of damage to the bone marrow microenvironment. Disruption of the hematopoietic microenvironment is indicated by delays in functional recovery of the immune system in spite of delivery of healthy peripheral blood or bone marrow progenitor cells. Cultures of primary human bone marrow stromal cells and a cloned murine stromal cell line, S10, were evaluated for functional changes following in vitro exposure to the chemotherapeutic agent, etoposide (VP-16). Stromal cells had reduced capacity to support lymphoid or myeloid cell proliferation following chemotherapy treatment. A consistent reduction of vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stromal cells also followed VP-16 exposure. These observations indicate that functional disruption of the bone marrow microenvironment by chemotherapeutic regimens must be considered when attempting to optimize patient recovery from hematopoietic transplantation.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Stromal cells regulate bcl-2 and bax expression in pro-B cells.

B lymphocyte production in the bone marrow depends on a cascade of regulatory cells and cytokines unique to the hematopoietic microenvironment. Fibroblastic stromal cells appear to be particularly important in regulating the earliest events in this lineage; however, it is still not clear whether the same or different sets of signals regulate maintenance of cell viability, proliferation, and differentiation of B lineage cells. In this study, we addressed the role of bone marrow stromal cells in survival and expansion of normal murine pro-B cells. Stromal cells were required for long-term proliferation of pro-B cell clone C1.92, and, in the presence of stromal cell line S10, pro-B cells expressed the proto-oncogene bcl-2. Removal of C1.92 cells from Stromal cell-derived signaling in support of pro-B cell viability. Due to its previously described role in regulating cell survival, we investigated whether stromal cells regulate bcl-2 expression in pro-B cells. When removed from stromal cell cultures, pro-B cells rapidly lost bcl-2 mRNA expression coincident with initiation of apoptosis. However, interruption of bcl-2 expression with antisense oligonucleotides in the presence of stroma and interleukin-7 (IL-7) did not result in immediate cell death. Oligonucleotide-treated cells arrested in G(1) phase of the cell cycle 24 hours before the initiation of apoptosis. In contrast, removal of pro-B cells from stromal cell support resulted in rapid increase in BAX expression, correlating directly with initiation of apoptosis. These results suggest that bcl-2 may, in part, regulate cell survival by interrupting the cascade of intracellular events that regulate cell cycle progression in lymphopoietic cells. Initiation of apoptosis in these cells appears to be more closely correlated with intracellular levels of BAX expression.

Animals↗
PubMed1994

Selective myelotoxicity of propanil.

Propanil, a commonly used herbicide, has been previously shown to be immunotoxic for selected immune functions as well as specific cell types, such as the macrophage. Propanil has also been shown to cause a methemoglobulinemia and anemia through direct action on the erythrocyte. Demonstrated toxicity to both macrophages and erythrocytes raised concern for the possible myelotoxicity of propanil which could contribute to the observed effects of exposure. Therefore, the effect of propanil on several stem and progenitor cell types was assessed 7 days after acute propanil exposure. The results described herein show that propanil, at doses of 50-200 mg/kg body wt, resulted in reduction in the number of myeloid stem cells and early myeloid and erythroid progenitor cells. No reduction in the numbers of more differentiated myeloid and erythroid progenitor cells was noted at even the highest dose used (200 mg/kg). In addition, no statistically significant difference in number of leukocytes per femur was noted. These data suggest that propanil is myelotoxic to early hemapoietic stem cells, but that this reduction is apparently compensated by proliferation of more differentiated progenitor cells for the myeloid and erythroid lineages. It remains unknown whether chronic exposure leads to progressive depletion of additional myeloid and erythroid cells.

Animals↗
PubMed1994

Gender-specific effects of prenatal chlordane exposure on myeloid cell development.

Work previously reported by this laboratory indicated that prenatal chlordane exposure affected macrophage function in young adult mice. Because these macrophage effects were due to exposure during the development of the immune system, the possibility of a persistent effect on the development of myeloid stem and progenitor cells was considered. Female mice were treated with either 0 or 8 mg of chlordane per kilogram body weight daily for 18 days during pregnancy. Myeloid hemopoietic activity of bone marrow cells from 6-week-old offspring was evaluated for in vitro colony-forming units-in-culture in response to exogeneously added recombinant forms of the cytokines granulocyte/macrophage-colony stimulating factor, macrophage-CSF, and interleukin 3 (IL-3). There was a significant depression of the numbers of bone marrow colony forming units-granulocyte/macrophage (CFU-GM), CFU-IL-3, and CFU-macrophage (CFU-M) in only the female offspring. Male offspring consistently demonstrated no difference in the CFU-GM, CFU-IL-3, or CFU-M. Prenatal treatment with chlordane did not significantly affect the number of recoverable viable bone marrow cells in either male or female mice.

Animals↗
PubMed1994

Differential expression of bone marrow stromal cell-surface antigens on myeloid and lymphoid cells.

Two monoclonal antibodies (MAb) that recognize cell-surface determinants present on bone marrow stromal cells have been generated. The 5B3 antibody recognizes a major histocompatibility class I-like molecule. This antigen is coexpressed on all bone marrow B-lineage cells and myeloid progenitors responsive to recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). The 9D3 antibody recognizes a protein similar in size to those encoded by the Ly-6 locus. A comparable bone marrow staining pattern and inhibition of 9D3 binding to bone marrow cells by anti-Ly-6C antibodies strongly suggests that the 9D3 antigen is Ly-6C. These data provide a further phenotypic characterization of bone marrow stroma and indicate that these cells express cell-surface determinants also present on myeloid and lymphoid cells.

Animals↗
PubMed1993

Insulin-like growth factor-1 potentiates expansion of interleukin-7-dependent pro-B cells.

Commitment to B-lymphocyte differentiation is characterized by expression of the B220 form of the common leukocyte antigen (Ly-5) and D-JH rearrangement of the Ig heavy chain gene complex. B-lineage progenitor cells, or pro-B cells, that have initiated Ig gene rearrangement, but do not express detectable Ig heavy or light chain protein, have recently been shown to retain substantial capacity for expansion in vitro in the presence of bone marrow (BM) stromal cells and interleukin-7 (IL-7). Although the potentiating effect of stromal cells on pro-B-cell proliferation can be partially attributed to the ligand for the proto-oncogene receptor c-kit (c-kit ligand [KL] or stem cell factor), several lines of evidence suggest that c-kit-mediated cell signalling is not required for pro-B-cell expansion. Previous studies from this laboratory demonstrated that insulin-like growth factor-1 (IGF-1) potentiated the proliferative effect of IL-7 on nonadherent cells from lymphoid long-term BM cultures in a manner similar to that shown for KL. To further delineate specific cell stages that respond to lymphopoietic cytokines, we derived continuously proliferating pro-B-cell lines from day-14 murine fetal liver in the presence of IL-7 and BM stromal cell clone S10. Initial expansion and continued proliferation of these pro-B-cell lines was absolutely dependent on the presence of both IL-7 and stromal cells. In the absence of KL, IL-7-stimulated proliferation of these cells in short-term cultures and addition of either recombinant IGF-1 or KL significantly potentiated this proliferative response. Although IGF-2 and insulin also potentiated the effect of IL-7, our data suggest that neither IGF-2 nor insulin represent normal regulators of intramyeloid lymphocyte development. IGF-1 and KL activate unique cascades of intracellular signalling events and inclusion of both cytokines in cultures of IL-7-stimulated pro-B cells resulted in additive potentiation of the proliferative response. Taken together, these results suggest that expansion of pro-B cells in vivo is maintained by at least three stromal cell-derived cytokines. IL-7 appears to be unique in delivering the primary proliferative signal for pro-B-cell expansion; however, both KL and IGF-1 potentiate the proliferative effect of IL-7 on these cells. The functional redundancy and additive effects of IGF-1 and KL as amplification signals for developing B-lineage cells underscore the essential nature of clonal expansion and diversification in development of immunocompetent lymphoid cells.

Animals↗
PubMed1992

Insulin-like growth factor-I regulates pro-B cell differentiation.

Progression of B-lymphocyte development in the bone marrow of postnatal mammals is marked by progressive rearrangement and expression of immunoglobulin (Ig) heavy- and light-chain genes. Following productive VHDJH gene rearrangement in the Ig heavy-chain gene complex, mu-heavy chain is the first Ig gene product expressed in cells committed to the B-lymphoid differentiation pathway. Interleukin (IL)-7 has been shown to stimulate proliferation of pre-B cells following c mu expression and this proliferative stimulus is potentiated by kit ligand (KL). However, it appears that neither of these cytokines contributes to differentiation of pro-B cells or initiation of expression of Ig gene products. We previously demonstrated that differentiation of pro-B cells and expression of mu-heavy chain is stimulated by either bone marrow stromal cell line S17 or cell-free supernatants from that line. This biological activity was attributed to molecules with an apparent M(r) of less than 10 Kd and approximately 40 to 60 Kd. We now report that this biological activity resides with stromal cell-derived insulin-like growth factor-I (IGF-I). Recombinant IGF-I stimulated the expression of cytoplasmic mu-heavy chain in short-term bone marrow cultures and this stimulus was abrogated in the presence of anti-IGF-I antibody. We also demonstrate that either anti-IGF-I antibody or pretreatment of S17 cells with antisense oligonucleotide for IGF-I abrogated the pro-B cell differentiation activity of S17 stromal cell supernatants. Although IGF-I did not directly stimulate proliferation of B-lineage cells, like KL, it potentiated the proliferative stimulus provided by IL-7. Taken together, these data strongly suggest that IGF-I produced by bone marrow stromal cells in the hematopoietic microenvironment plays a key role in regulating primary B lymphopoiesis.

Animals↗
PubMed1992

Differential roles of stromal cells, interleukin-7, and kit-ligand in the regulation of B lymphopoiesis.

Newly formed B lymphocytes are a population of rapidly renewed cells in the bone marrow of mammals and their steady state production presumably depends on a cascade of regulatory cells and cytokines. Although considerable information has been forthcoming about the role of interleukin-7 (IL-7) in potentiating pre-B-cell proliferation, few studies have addressed the possibility that multiple cytokines are involved in the progression of early events in cellular differentiation and proliferation in this hematopoietic lineage. Our laboratory previously described pre-B-cell differentiation mediated by the bone marrow stromal cell line S17. In this study, we further delineate the role of stromal cells in differentiation and proliferation of pre-B cells. These experiments show that the stromal cell line S17 potentiates the proliferative effect of IL-7 on B-lineage cells and that this S17-derived potentiator can be replaced with recombinant kit-ligand (KL). Our results further show that pre-B-cell formation from B220-, Ig- progenitor cells and expression of mu heavy chain of immunoglobulin is uniquely dependent on the presence of S17 stromal cells and cannot be reproduced with IL-7, KL, or costimulation with both IL-7 and KL. These data contribute to a rapidly evolving model of stromal cell regulation of B-cell production in the marrow and suggest unique roles for IL-7, KL, and as yet uncharacterized stromal cell-derived lymphokines in this process.

Animals↗
PubMed1992

Regulation of B cell differentiation by bone marrow stromal cells.

B lymphocyte development occurs in the intersinusoidal spaces of bone marrow in association with a sessile population of stromal cells. Development of long-term bone marrow culture systems that support B lymphopoiesis has allowed the isolation of stromal cells that support that process and permitted analysis of their role in controlling the growth and differentiation of B lineage cells in culture. In addition to direct interactions with developing lymphocytes, stromal cells secrete a variety of cytokines that affect lymphocyte growth and differentiation, and many of these molecules have been expressed in recombinant form. These relatively recent advances have made it possible to formulate a working model of the cells and molecules involved in regulating primary B cell production.

Animals↗
PubMed1992

Regulatory cells and cytokines involved in primary B lymphocyte production.

Based upon the above data, it is now possible to formulate a working model that defines the stages of B cell development on which stromal cells and their products act. During the initial stages of this process, pro-B cells which do not express Ig heavy or light chain protein or other non-Ig B lineage associated molecules develop into B220 and c mu expressing pre-B cells in response to a low (< 10 kD) molecular weight stromal cell derived factor. No defined interleukin or colony stimulating factor, including molecules such as KL and IL-7, can replace stromal cell conditioned medium in mediating this developmental step. There appears to be little cell proliferation associated with the differentiation of pro-B cells into pre-B cells. However, our data indicate that as precursors develop into B220 expressing B cell progenitors, they become sensitive to the proliferation stimulating effects of IL-7 and KL. These results are in accord with findings that progenitor cells that have undergone DJH rearrangements are particularly sensitive to KL and IL-7(18,19). The analysis of pre-B cells present in individual lymphoid colonies indicates that once cells have rearranged and expressed their Ig heavy chain genes, they are no longer sensitive to KL and IL-7. These observations are based on the fact that receptors for these cytokines are not expressed in stromal cell dependent pre-B cells and are consistent with kinetic studies showing that the maturation of pre-B cells into surface Ig expressing B lymphocytes is not dependent upon cell proliferation21.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocyte Subsets↗
PubMed1990

Bone marrow stromal cell regulation of B lymphopoiesis: interleukin-1 (IL-1) and IL-4 regulate stromal cell support of pre-B cell production in vitro.

Bone marrow stromal cells appear to be key regulatory elements in hematopoiesis and lymphopoiesis. These stromal cells respond to cytokine exposure and alter their pattern of hematopoietic growth factor production, suggesting a degree of functional plasticity. We examined the effect of two cytokines, interleukin-1 (IL-1) and IL-4, on stromal cell regulation of pre-B cell generation using the bone marrow stromal cell line, S17. Neither lymphokine potentiated pre-B cell generation in the absence of stromal cells. However, addition of either 10 U/mL rIL-1 alpha or 50 U/mL rIL-4 to cultures of bone marrow cells containing S17 cells dramatically suppressed subsequent pre-B cell formation. Preculture of S17 stromal cells with either rIL-1 or rIL-4 completely abrogated their ability to support pre-B cell generation in subsequent coculture with freshly explanted bone marrow cells. Conditioned medium from IL-1- or IL-4-treated S17 cells also suppressed pre-B-cell generation in culture. Although it is not yet known which induced stromal cell factors are responsible for failure of pre-B-cell generation in treated cultures, these data do clearly demonstrate that local levels of IL-1 and IL-4 in the hematopoietic microenvironment may play a significant role in regulation of bone marrow stromal cell function. These data also demonstrate that fibroblastic stromal cells are primary target cells that respond to cytokine concentration and affect lymphopoietic cell development.

Animals↗
PubMed1989

A colony assay system that detects B cell progenitors in fresh and cultured bone marrow.

This report describes a colony assay system, based on methods used to grow myeloid precursors in semisolid medium, in which B cell progenitors can be grown. The formation of these B cell progenitor colonies is dependent upon soluble mediators from a stromal cell line known to support B lymphopoiesis. In initial experiments a double layer culture system was employed in which target cells in methylcellulose medium were separated from an adherent layer of S17 stromal cells by an agar interface. Target cells were harvested from Dexter type long-term bone marrow cultures at a time after transfer to the lymphoid Whitlock-Witte conditions, when myeloid progenitors were depleted and mature B cells had not yet appeared. On day 15 of culture a colony could be identified that contained several hundred tightly clustered lymphoid cells. There was a linear relationship between the number of cells plated and the number of colonies that developed. Identically appearing colonies were also observed in agar using fresh bone marrow cells as targets with either an underlayer of S17 cells or S17 conditioned medium to potentiate colony growth. Lymphoid colonies derived from fresh bone marrow appeared on days 6 and 14 of growth. A proportion of the cells from the fresh or cultured marrow derived colonies expressed the B220 antigen and cytoplasmic mu heavy chains, but surface IgM was never observed. Cell depletion experiments on antibody coated plates demonstrated the colony forming unit to be B220 antigen positive, surface IgM negative, and replating experiments indicated the colonies were lymphoid restricted in their differentiative potential.

Animals↗