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C W Reynolds

Publications and source records attributed to C W Reynolds.

At least 37 records · Page 2Linked to original sources
PubMed1990

Differential regulation of interleukin-1 gene expression in human CD3- large granular lymphocytes.

Our laboratory analyzed the expression of lymphokine and cytokine mRNA in CD3- peripheral blood large granular lymphocytes (LGL). Herein we present evidence that this subset of lymphocytes can synthesize IL-1 beta mRNA constitutively and that the cytoplasmic mRNA levels of IL-1 beta can be increased rapidly by interleukin (IL)-2. IL-1 alpha mRNA is expressed constitutively very infrequently and increases in IL-1 alpha mRNA are seen only after prolonged incubation with IL-2. Furthermore, IL-1 activity could not be detected in LGL culture supernatants, indicating that other processes may be involved in releasing biologically active IL-1 from LGL. In addition, MAb to the p75 IL-2 receptor on LGL abrogated IL-2 induction of IL-1 beta mRNA, suggesting that IL-2 signaling via the p75 IL-2 receptor induced IL-1 beta gene expression in LGL. Since, in contrast to T cells, LGL are capable of mediating effector functions without prior stimulation, they are said to be already "primed" for response. Overall, these data suggest that constitutive lymphokine gene expression may be involved in the in vivo priming of LGL.

Antigens, CD↗
PubMed1990

Intrinsic cytotoxicity of natural killer cells to pancreatic islets in vitro.

BB rats develop spontaneous autoimmune insulin-dependent diabetes mellitus that is similar to human insulin-dependent diabetes. In this study, we used an in vitro islet cell cytotoxicity assay to study the possible role of natural killer (NK) cells and their soluble effector molecules in this disorder. First, the results demonstrated that in vivo treatment of acutely diabetic BB rats with anti-asialogangliosideM1 (an NK cell antiserum) but not with anti-T-lymphocyte antibodies reduces spleen cell cytotoxic activity to islets in vitro. Flow microfluorometry (FMF)-sorting experiments were then used to confirm that the splenic cytotoxic effector cell in acutely diabetic BB rats is a CD8+/CD5- NK cell. Further analysis demonstrated that both FMF-sorted NK cell populations from Wistar-Furth rats and unfractionated spleen cells from athymic nu/nu rats with high intrinsic NK cell activity also exhibit high islet cell cytotoxic activity in vitro. Finally, we found that the kinetics and differential cytotoxic activity of NK cells toward islets in vitro could be mimicked by NK cell culture supernatants containing high levels of NK cytotoxic factor (NKCF). The islet cytotoxic activity of these culture supernatants was specifically inhibited by the addition of anti-NKCF monoclonal antibody. These results demonstrate that NK cells from diabetic and nondiabetic rats are cytotoxic to islet cells in vitro. They further suggest that this cytotoxic effect may be mediated in part through the production and release of soluble factors such as NKCF.

Animals↗
PubMed1989

Molecular cloning of rat cytolysin.

Rat cytolysin is one of the cytolytic factors present in the cytoplasmic granules of rat NK-like cytolytic cells and purified cytolysin exhibits an apparent Mr or 70 kDa. Cytolysis produced by cytolysin occurs in the presence of Ca2+ and is accompanied by the formation of membrane lesions of 160 A diameter. We have isolated a cDNA encoding rat cytolysin from the cDNA library of a rat large granular lymphocyte (LGL) cell line, by hybridization of the rat library with a cDNA probe for mouse perforin. The amino acid sequence deduced from the nucleotide sequence of the isolated cDNA insert indicates that the mature cytolysin protein consist of 534 amino acids with a leader peptide of 20 amino acids. The protein contains two functionally important domains: the first domain is believed to contain the transmembrane channel and the second domain consists of an epidermal growth factor-type "class B" cysteine-rich region. A comparison with mouse perforin indicates that the two genes are very similar (89.9% nucleotide and 84.9% amino acid identity). Northern blot hybridization analysis indicates that cytolysin mRNA is expressed in rat lymphocytes (lymphokine-activated killer cells and LGL cells) and LGL cell lines.

Amino Acid Sequence↗
PubMed1989

Identification and characterization of a cell-surface molecule that is selectively induced on rat lymphokine-activated killer cells.

We have identified a 40- to 45-kDa cell-surface molecule designated gp42, that is expressed in high levels by rat lymphokine-activated killer (LAK) cells of NK cell origin. gp42 cannot be detected on the precursors of LAK cells and is not present on resting or activated T cells. Rather, expression of gp42 is selectively induced on NK cells by the high concentrations of rIL-2 that are required for the induction of LAK activity. Although the function of gp42 is not known, the selective nature of its expression suggests a role for this molecule in regulating responses that are unique to IL-2-activated NK cells.

Animals↗
PubMed1989

Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow.

A variety of biologic response modifiers (BRM) can potently augment NK activity in nonlymphoid organs. By using the liver as a model organ, we have shown that this augmentation of organ-associated NK activity is coincident with a 10- to 15-fold increase in the number of large granular lymphocytes (LGL) which can be isolated. The present study was designed to investigate the mechanism by which BRM induce this increase in liver-associated LGL and the coincident increase in hepatic NK activity. Initial studies confirm that a single dose of the pyran copolymer, maleic anhydride divinyl ether (MVE-2), augmented hepatic NK activity and increased the number of liver-associated LGL from 3 x 10(4)/liver to 5 x 10(5)/liver (a 17-fold increase). Multiple injections of MVE-2 further augmented total liver-associated NK activity and LGL number (to 13 x 10(5)/liver). As expected, both the NK activity and detectable LGL were eliminated by treatment of the mice with antiasialo GM1 (asGM1) serum. Three possible mechanisms for the BRM-induced increase in liver-associated LGL have been investigated, including 1) the rapid proliferation of resident hepatic LGL, 2) the redistribution of mature LGL from peripheral sites such as the spleen, or by 3) a rapid output and subsequent hepatic localization of LGL or their precursors recently derived from the bone marrow (BM). Our results demonstrated that the contribution of in situ proliferation to the BRM-induced increase in liver-LGL was relatively small, since the number of cells expressing NK-associated markers (i.e., asGM1, Thy-1.2, and NK1.1) and in G2/M phase (as assessed by propidium iodide uptake) was only 4 to 8%. Further experiments demonstrated that splenectomy before the administration of MVE-2 did not inhibit the augmentation of liver-associated NK activity. This result argued against a recruitment of mature LGL from the spleen. In contrast, selective depletion of the BM following administration of 89Sr decreased the ability of MVE-2 to augment liver-associated NK activity by greater than 80%. This procedure also significantly decreased the ability of Propionibacterium acnes (85%) and multiple doses of IL-2 (49%) to augment liver-associated NK activity. These results demonstrate that the rapid augmentation of liver-associated NK activity by BRM is largely due to localization and accumulation in the liver of LGL recently derived from the BM.

Adjuvants, Immunologic↗
PubMed1989

The proliferation and life-span of rat large granular lymphocytes: effects of cytokines.

Pulse labeling of large granular lymphocytes (LGL) with [3H]thymidine for 1 h in vitro showed that 1%-7% of LGL were in S phase in the blood, spleen and liver of unstimulated euthymic and athymic rats as scored with autoradiography. Repetitive injections of [3H]thymidine over 2-7 days revealed that about half of the blood and spleen LGL had formed by division of precursors during this week. Stimulation of rats with the interferon inducer poly(I).poly(C) increased the proportion of S phase LGL rapidly and simultaneously in the blood, spleen and liver, so that by 20 h after stimulation ca 30% of LGL were in the S phase in these organs. This LGL proliferation was accompanied by an increased number of LGL in all three compartments 48-96 h after poly(I).poly(C) injection. Blood LGL cultured in cell impermeable diffusion chambers in the peritoneal cavity of poly(I).poly(C)-stimulated rats exhibited enhanced natural killer (NK) activity but no proliferative response, indicating that mature LGL were not induced to undergo blastogenesis by poly(I).poly(C) in vivo. Pretreatment of NK cells with rat interferon (IFN-alpha/beta) and with human recombinant interleukin 2 (rIL 2) in vitro, showed that these two cytokines, when combined, had opposing effects on NK activity and proliferation: whereas rIL 2 inhibited the IFN-induced augmentation in NK activity, IFN inhibited the rIL 2-induced LGL proliferation.

Animals↗
PubMed1989

Mechanism of immunotherapeutic activity of OK-432 in the treatment of peritoneal carcinomatosis.

In this report the mechanism of therapeutic activity of OK-432 for the treatment of peritoneal carcinomatosis was investigated by correlating effector-cell augmentation with therapeutic activity in rats bearing MADB-106 carcinomatosis. Tumor cells were injected i.p. and the treatment with OK-432 was initiated 5 days later with 0.5, 1, 5 or 10 KE/animal of OK-432 injected i.p. semiweekly. Significant therapeutic activity was observed at all doses examined with greater prolongation of survival noted at the higher doses of OK-432. Animals treated with 0.5 KE/animal had a prolongation of the median survival time from 14 days for saline-treated animals to 17 days for the OK-432 treated animals (P less than 0.0008), while animals treated with higher doses had much longer periods of survival, some animals being tumor-free at 185 days. In the same studies, natural killer (NK) cell, lymphokine-activated killer cell, cytotoxic T lymphocyte, and macrophage tumoricidal/cytostatic activities were measured 7 days and 14 days following tumor injection (2 days and 9 days after initiation of immunotherapy). OK-432 had immunostimulatory activity in most of the assays of immune function examined and this correlated with host survival, including augmentation of peritoneal and peripheral blood cytotoxic T lymphocyte activity on day 14, peritoneal and alveolar macrophage activity on day 7 and day 14, as well as natural killer cell activity on day 14. These results suggest that the therapeutic doses are also immunomodulatory doses for the effector cells mentioned above. We suggest, therefore, that immunological monitoring may help to optimize treatment protocols for the treatment of peritoneal and perhaps pleural effusions with OK-432.

Adjuvants, Immunologic↗
PubMed1988

IFN-gamma treatment of K562 cells inhibits natural killer cell triggering and decreases the susceptibility to lysis by cytoplasmic granules from large granular lymphocytes.

Pretreatment of human K562 leukemia cells with rIFN-alpha and rIFN-gamma resulted in decreased susceptibility to lysis by human peripheral blood NK cells. The reduction of NK-susceptibility after IFN treatment was not due to a general effect of IFN on the stability of the cell membrane because the susceptibility of K562 cells to lysis by antibodies plus C, distilled water, or lysolecithin was unaffected. Binding studies with effector cell preparations enriched for NK cells with large granular lymphocyte morphology revealed no difference in binding to control and IFN-gamma-treated target cells. The sensitivity to soluble NK cytotoxic factors was not affected significantly by the IFN treatment. In contrast, the susceptibility of IFN-treated target cells to the cytotoxic activity of purified cytoplasmic granules from a rat large granular lymphocyte tumor was significantly reduced, indicating that the IFN-induced resistance acted at the level of susceptibility to the lytic mechanism of NK cells. However, IFN-alpha was more effective than IFN-gamma in inducing resistance to the cytoplasmic granules although resulting in only a weak resistance in the cell-mediated cytotoxic assay. IFN-gamma but not IFN-alpha caused a reduction in the frequency of effector cells that had reoriented their Golgi apparatus toward their bound target cell. In addition, IFN-gamma treated K562 cells failed to elicit an influx of Ca2+ into effector cells. Taken together, the results suggest that IFN-gamma in addition to an increased resistance to the lytic molecules released by NK cells can also induce changes in the target cells which prevent the triggering and activation of the effector cell.

Animals↗
PubMed1988

Lymphokine-activated killer cells in rats: analysis of progenitor and effector cell phenotype and relationship to natural killer cells.

The progenitor and effector cell phenotype of lymphokine-activated killer (LAK) cells generated in F344 rats by recombinant human interleukin 2 (IL-2) (rIL-2) were analyzed. Highly purified populations of peripheral blood large granular lymphocytes (LGL) exhaustively depleted of T-cells were fully capable of generating high levels of LAK activity by 3 to 5 days in culture while purified populations of resting T-cells devoid of LGL could not generate LAK activity. This pure population of LGL expressed surface markers characteristic of rat natural killer (NK) cells [i.e., OX8+, asialomonoganglioside (asialo-GM1+), laminin+, OX19-, R1-3B3-, W3/25-, Ia-, surface immunoglobulin negative (SIg-)]. Further evidence that NK cells were the progenitors of cells with LAK activity was obtained by treatment of spleen or peripheral blood lymphocytes with anti-laminin or anti-asialo-GM1 antibodies plus complement or with the lysosomotropic agent L-leucine methyl ester. These treatments effectively depleted LGL/NK cell activity and the subsequent generation of rIL-2-induced LAK activity. Analysis of the LAK effector phenotype by cell sorting demonstrated that the majority of cells with LAK activity were OX8+, asialo-GM1+, laminin+, OX6+, OX19-, R1-3B3-, W3/25-, and SIg-. Furthermore, treatment of LAK cells with L-leucine methyl ester also significantly reduced their cytolytic activity. Thus, the LAK effector cells were also LGL and expressed surface marker characteristic of activated NK cells and not those of mature T- or B-cells. The proliferative response of rat spleen or blood lymphocytes to rIL-2 appeared to be primarily associated with LGL/NK cells since depletion of NK cells by anti-asialo-GM1 or anti-laminin antibody plus complement or by L-leucine methyl ester significantly (P less than 0.001) reduced the incorporation of [3H]thymidine into DNA. In contrast, depletion of T-cells (by anti-T-cell antibody plus complement) did not significantly affect rIL-2-induced proliferation. Similarly, T-cell-depleted, highly purified populations of LGL gave substantial proliferative responses to rIL-2. These studies clearly indicate that in the rat, the major cell population activated by rIL-2 is the LGL/NK cell and these cells appear to represent the major population of cells in blood or spleen which generate broad antitumor (LAK) cytotoxicity.

Animals↗
PubMed1988

Features of the in vitro established rat large granular lymphocyte leukaemia RNK-16.

A large granular lymphocyte (LGL) leukaemia cell line from the Fisher/F344 rat strain called RNK-16 has been established in vitro, maintaining the same surface markers as tumour cell growing in vivo. The tumour has also maintained its specificity pattern and cytotoxic reactivity and serves as a suitable source of natural killer (NK)-like effector cells in vitro. The cells show no evidence of dependency on, or production of, interleukin 2 or interferons, nor is the cytotoxic capacity influenced by treatment with mitogens. The in vitro line does not produce natural killer cytotoxic factor (NKCF) in a constitutive manner, but can be induced to do so via coculture with tumour target cells. When the fine specificity patterns were analysed, the RNK-16 cells express species-preferential lysis of susceptible target cells and a highly discriminatory power to kill only 1 out of 5 rat erythroleukaemia cell lines. When testing normal target susceptibility patterns, RNK-16 kills lymphoblasts of B type better than T blasts, which is well in line with previous findings on normal NK cell specificity patterns.

Animals↗
PubMed1987

Analysis of rat natural killer cytotoxic factor (NKCF) produced by rat NK cell lines and the production of a murine monoclonal antibody that neutralizes NKCF.

Natural killer cytotoxic factor (NKCF) is produced as a result of the interaction of murine, rat, or human natural killer (NK) cells with NK-susceptible targets. This factor has been linked to the target cell lysis mediated by the NK effector cell. In the present results, culture supernatants from rat large granular lymphocyte (LGL) tumors exhibited NKCF activity which lysed the susceptible targets, MBL-2 and YAC-1. NKCF production from these rat tumor lines was spontaneous and was not significantly increased by co-incubation of the LGL tumors with target cells, target cell membranes, or by preincubation of the LGL tumor cells with interferon or interleukin 2. In addition to NKCF activity, the supernatants lysed L929, indicating the presence of tumor necrosis factor (TNF) in these preparations. The presence of this latter cytokine was verified using specific antibodies to recombinant murine TNF which neutralized the L929 activity while not affecting the NKCF activity against MBL-2 or YAC-1. Mouse monoclonal antibodies (mAb) A0287, A0462, and A0316) which significantly inhibit the NKCF cytolytic activity of these LGL-derived supernatants were also produced. These antibodies were shown to cross-react with human NKCF in a manner similar to that seen in the rat. Interestingly these same mAb demonstrated no inhibition of L929 cytotoxicity from either LGL-derived supernatants or by recombinant murine or human TNF. To examine further the specificity of these antibodies, they were chemically linked to Sepharose 4B and found to remove a significant proportion of the NKCF cytolytic activity from LGL supernatants, while not affecting the TNF reactivities in these preparations. In addition, these antibodies demonstrated significant inhibition of cell-mediated cytotoxicity by rat LGL against YAC-1 target cells. Biochemical analysis of labeled NKCF-containing supernatants indicated the major protein recognized by these anti-NKCF mAb to be approximately 12,000 m.w. The use of these mAb against NKCF should be very useful in further purification and biochemical characterization of NKCF and in studying its role in a variety of cell-mediated cytotoxicity assays.

Animals↗
PubMed1987

Co-sedimentation of chondroitin sulfate A glycosaminoglycans and proteoglycans with the cytolytic secretory granules of rat large granular lymphocyte (LGL) tumor cells, and identification of a mRNA in normal and transformed LGL that encodes proteoglycans.

Rat large granular lymphocyte (LGL) tumor cell lines were analyzed for the presence of proteoglycans and glycosaminoglycans in their cytolytic secretory granules. When isolated rat LGL tumor cells were incubated in vitro for 1 to 3 hr with [35S]sulfate, and the 35S-labeled macromolecules were purified by density-gradient centrifugation, they filtered on Sepharose CL-4B columns predominantly as approximately 500,000 m.w. macromolecules. After 19 hr of incubation with [35S]sulfate, however, an 85,000 m.w. species predominated. Pulse-chase experiments revealed that the larger macromolecules were proteoglycans that with time were processed to glycosaminoglycan-sized macromolecules. As assessed by their susceptibility to chemical and enzymatic degradation and by high pressure liquid chromatography of the chondroitinase ABC-generated unsaturated disaccharides, the cell-associated rat LGL tumor cell proteoglycans bore almost exclusively chondroitin sulfate A glycosaminoglycans. Northern blot analysis using a gene-specific probe revealed that both normal peripheral blood and transformed rat LGL expressed the same approximately 1.3-kb mRNA that encodes the peptide core of the proteoglycans in the secretory granules of rat and mouse mast cells. In vivo radiolabeling of rat LGL tumor cells and isolation of their intact granules after nitrogen cavitation and density sedimentation established that glycosaminoglycans compartmentalized with cytolytic activity. Thus these negatively charged macromolecules may play a role in the regulation of the packaging and delivery of the cytolysins and basically charged serine proteases that have been identified in the cytolytic secretory granules of LGL.

Aggrecans↗
PubMed1987

Natural killer cell activity in the rat. VI. Characterization of rat large granular lymphocytes as effector cells in natural killer and antibody-dependent cellular cytotoxic activities.

The present study examined rat natural killer (NK) cells, which mediate not only NK activity but also antibody-dependent cellular cytotoxicity (ADCC). NK and ADCC activities were compared with regard to organ distribution, strain distribution, Percoll fractionation of the effector cells, effects of aging, and potential to be augmented by biological response modifiers (BRM). Like NK activity, appreciable ADCC activity was observed in peripheral blood leukocytes (PBL), splenic leukocytes (SPL), and peritoneal exudate cells (PEC), but not in cell preparations from the peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), bone marrow (BM), and thymus (THY). ADCC activity, when compared with NK activity, was significantly higher in PBL but the same or lower in SPL and PEC. In terms of strain distribution, a high NK/ADCC strain (rnu/rnu), four intermediate NK and high ADCC strains (PVG/RTLRL, Lewis, PVG/OLA, and F344), an intermediate NK/ADCC strain (WF/N), and a low NK/ADCC strain (Buffalo) were observed. Fractionation of effector cells on discontinuous Percoll gradients revealed that both NK and ADCC activities were associated with relatively high-density large granular lymphocytes (LGL). In contrast, ADCC but little or no NK activity was associated with lower density LGL. However, the NK activity of this lower-density LGL population could be elicited following the in vitro incubation with a number of BRM, including rat interferon (IFN) and OK-432, but not rat interleukin-2 (IL-2). In general, the ADCC activity of both higher and lower density LGL-enriched cell populations correlated with both the frequency of FC gamma R+ LGL and the percentage of LGL binders to antibody-coated P815 target cells. The present study also has shown that in contrast to NK activity, which remained relatively stable with age, ADCC activity from F344 but not WF/N rats increased until 30-50 wk of age. This increase of ADCC activity in older F344 rats was accompanied by an increase in the percentage and absolute number of lower density FC gamma R+ LGL. This study demonstrates a number of similarities and differences between NK and ADCC activities in the rat. These findings should be useful for further examining and comparing the in vivo development and biological role of these two effector arms of the immune system.

Aging↗
PubMed1987

Inhibition of NK and ADCC activity by antibodies against purified cytoplasmic granules from rat LGL tumors.

Highly purified preparations of cytoplasmic granules from transplantable rat large granular lymphocyte (LGL) tumor lines (rat natural killer (RNK) tumors) were used to immunize rabbits. Antibodies from these animals gave two precipitin lines with granule extracts in Ouchterlony experiments. They reacted with at least four different bands on nitrocellulose blots of SDS gels of LGL granule proteins. By immunofluorescence, specifically adsorbed antigranule antibodies did not recognize LGL or T cell surface antigens but reacted with the cytoplasmic granules in permeabilized RNK tumor cells as well as with normal rat LGL. These same antisera showed little or no reactivity with a panel of other cells, including peripheral blood T cells, thymocytes, macrophages, and EL-4 tumor cells. F(ab')2 preparations of these antigranule antibodies completely blocked granule-mediated lysis of both SRBC and nucleated targets, while control F(ab')2 preparations from rabbits immunized with EL-4 granules or TNP-KLH showed no significant inhibition of this cytolytic activity at the same antibody concentration. Anti-granule F(ab')2 preparations specifically inhibited (greater than 75%) rat natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in a dose-dependent manner but did not effect cytotoxic T cell activity. Pretreatment of either effectors or targets by these antibodies had no effect. Anti-granule F(ab')2 preparations, at concentrations showing strong inhibition of lysis, did not inhibit the binding of LGL to YAC-1 or Ab-coated P815 targets. These results demonstrate that a granule component(s) is necessary for the lytic activity of LGL in both NK and ADCC and provide the first direct evidence that a secretory event involving these granules is part of the lytic process.

Animals↗
PubMed1987

Inhibition of cytolysin activity in large granular lymphocyte granules by lipids: evidence for a membrane insertion mechanism of lysis.

The ability of various compounds to inhibit the lytic activity of purified cytoplasmic granules from LGL tumors was tested. The lytic activity of granule cytolysin was modestly inhibited by inorganic phosphate, and various monophosphoesters, with I50 values in the range of 8-20 mM. Among monophosphoesters, choline phosphate was exceptionally potent, with an I50 of 1.4 mM. In contrast to the inhibition by phosphate esters, the parent compounds such as neutral sugars, glycerol, and choline, showed no detectable inhibition at 50 mM. A lysolipid bearing a short aliphatic chain and some detergents were inhibitory with I50 values in the range of 1 mM. Lysolipids with longer aliphatic chains, phospholipids as liposomes, and related lipid compounds were found to display potent inhibition of the hemolytic activity of LGL granule cytolysin, with I50 values in the range of 0.2-30 microM. Soluble globular proteins inhibited LGL granule cytolysin hemolytic activity with I50 of 0.07-0.4 mg/ml. However, lipoproteins were 2-3 orders of magnitude more potent inhibitors, with L50 values of less than 1 microgram/ml. The observed potent inhibition by lipid compounds lends support to a model of the lytic action of granule cytolysin in which soluble cytolysin molecules insert into the membrane lipid bilayer during the course of the lytic event.

Animals↗
PubMed1987

Inhibition of pluripotent hematopoietic stem cells of bone marrow by large granular lymphocytes.

Previous studies suggested that natural killer (NK) cells are involved in the regulation of the growth and differentiation of pluripotent hematopoietic stem cells. To establish whether the effector cells responsible for the in vivo resistance to bone marrow (BM) transplants and the in vitro inhibition of colony-forming units (CFU) may represent identical or overlapping populations, we used a rat system for syngeneic BM transplantation, with and without the transfer of large numbers of peripheral blood large granular lymphocytes (LGLs). BM reconstitution was measured by the in vivo formation of syngeneic CFU in the spleen (CFU-s). Because of the very low frequency of CFU-s in normal rat BM, we fractionated BM cells in Percoll density gradients, which provided a 2- to 5-fold enrichment in CFU-s in the lower-density fractions. Although these fractions contained less than 10% of the total cells, they contained greater than 75% of the CFU-s and allowed for the transfer of significantly fewer donor cells. At the time of BM transplantation, radiation-resistant asialoganglioside GM1-positive LGLs, with high NK activity, accounted for a significant percentage of the lymphoid cells in the irradiated recipient. The in vivo regulatory role of these cells on engraftment was demonstrated by their depletion (by i.v. injection of small amounts of anti-asialo-GM1 antiserum before BM transplantation), which resulted in a significant increase in the number of CFU-s. Conversely, a 50% inhibition in CFU-s was found when CFU-s-enriched BM fractions were preincubated in vitro with LGLs. Additional experiments, involving selective in vivo depletion of NK cells followed by LGL repopulation, directly demonstrated the involvement of LGLs in the regulation and growth of syngeneic pluripotent hematopoietic stem cells. Our results further support the hypothesis that LGLs are involved directly or via humoral factors in the homeostasis and regulation of hematopoietic stem cell growth and differentiation.

Animals↗