Biomedical subjects

H S Warren

Publications and source records attributed to H S Warren.

At least 37 records · Page 2Linked to original sources

PubMed1996

Analysis of the costimulatory role of IL-2 and IL-15 in initiating proliferation of resting (CD56dim) human NK cells.

IL-15 is a newly described cytokine produced by monocytes and other non-T cells that utilizes the IL-2R beta- and common gamma-chains, thereby stimulating many NK cell functions previously ascribed to IL-2. Thus, IL-15 may promote NK cell activity during innate immune responses, before the activation of T lymphocytes and subsequent production of IL-2. This study investigated the ability of rIL-15 to substitute for rIL-2 in initiating proliferation of resting human NK cells cocultured with various stimulator cells. NK cell proliferation could not be initiated with rIL-15 as the sole costimulatory cytokine. However, NK cell proliferation was initiated with rIL-15 and either rIL-10 or rIL-12, cytokines also produced by monocytes and other APC and implicated in innate immune responses. Individually, rIL-10, rIL-12, and rIL-15 are effective initiators of NK cell proliferation when combined with submitogenic concentrations of rIL-2, indicating their potential involvement in NK cell proliferation at early stages of an Ag-specific T cell immune response. NK cells proliferating in the different cytokine combinations or optimum concentrations of rIL-2 were indistinguishable in terms of phenotype and cytotoxic activity, but differed in whether they secreted IFN-gamma or IL-5. IFN-gamma was secreted in cultures containing rIL-12, whereas IL-5 secretion was dependent upon interaction of IL-2 with the high affinity IL-2R. These results support the notion that NK cell proliferation occurs at different phases of the immune response with the particular cytokine milieu influencing the repertoire of NK cell-secreted cytokines.

Adjuvants, Immunologic↗

PubMed1996

A carbohydrate structure associated with CD15 (Lewis x) on myeloid cells is a novel ligand for human CD2.

The T cell and NK cell adhesion molecule CD2 interacts with different ligands, viz, CD58, CD48, and CD59. Using a fluorescent multimeric construct of rCD2, we previously identified an additional CD2 ligand (CD2L) on the erythroleukemic cell line K562. CD2L bound to a different region of CD2 than known ligands and was N-glycosylation dependent. In this study we show that mAbs specific for the carbohydrate Ag Lewis x (CD15, Gal-beta 1-4 GlcNAc alpha 1-3Fuc) inhibit multimeric rCD2 binding to CD2L. CD2L is restricted in expression to myeloid cells, where it is co-expressed with CD58 on monocytes and is the dominant, if not sole, CD2 ligand on neutrophils. Sugar specificity studies show that CD2L is not CD15. Thus, whereas soluble Lewis x inhibits binding of CD15 mAb to K562 and neutrophils, binding of multimeric rCD2 is unaffected. Furthermore, multimeric rCD2 binding to K562 is inhibited by L-fucose and following treatment of K562 with an alpha 1-6 fucosidase, whereas these treatments do not inhibit the binding of CD15 mAb. Thus, it is likely that CD2L is a carbohydrate structure closely associated with, yet distinct from, CD15, which can be sterically blocked by CD15 mAb. Functional studies revealed that CD2L is probably an important CD2 ligand in the non-MHC-restricted NK cell killing of K562 target cells, since this activity was strongly inhibited by CD15 mAb. Collectively, this study indicates that a CD15 (Lewis x)-associated carbohydrate structure(s), which has previously been shown to be a selectin ligand, also may function as an important CD2 ligand on myeloid cells.

Antibodies, Monoclonal↗

PubMed1996

NK cell proliferation and inflammation.

Considerable progress has been made in recent years in understanding the biology of NK cells. NK cells are no longer 'null cells', but express an array of functionally important molecules with which they mediate and regulate their cytolytic activity and the cytokines they secrete. Activation and proliferation of NK cells in influenced by cytokines produced by activated monocytes (IL-15, IL-12, IL-10) and activated T cells (IL-2). This paper reviews the phenotype and effector functions of NK cells, their tissue distribution, and evidence that NK cells proliferate in vivo as part of productive or pathologic consequences of immunity.

Animals↗

PubMed1996

Cytokine expression by high-density human lymphocytes.

T lymphocytes spend much of the time as small non-cycling cells. To determine the pattern of cytokine expression in such resting cells, they were purified from human peripheral blood mononuclear cells (PBMC) on the basis of high buoyant density. The cells were stimulated and cytokine mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Expression of interleukin-2 (IL-2), IL-3 and interferon-gamma (IFN-gamma) was similar in high-density lymphocytes and in unfractionated PBMC. In contrast, the high-density lymphocytes expressed less IL-4 than PBMC, and little or no IL-5. Because a substantial minority of the high-density lymphocytes was CD45RO+, the presence of this marker was not an indicator of the ability to express IL-4 and IL-5. In the high-density lymphocytes, IFN-gamma expression was confined to the CD45RO+ fraction, whereas IL-2 was expressed by both CD45RO+ and CD45RO- subsets. To assess whether high-density lymphocytes could give rise to cells with a broader range of inducible cytokine expression, they were activated and then restimulated between 10 and 22 days of culture. Cells derived from both the CD45RO+ and CD45RO- fractions of high-density lymphocytes expressed IL-5 after restimulation. Thus the high-density lymphocyte population has the potential to acquire a broader range of inducible cytokine expression.

Base Sequence↗

PubMed1995

Production of IL-5 by human NK cells and regulation of IL-5 secretion by IL-4, IL-10, and IL-12.

Human NK cells produce IFN-gamma, TNF-alpha, and granulocyte macrophage-CSF when stimulated with susceptible target cells or through the CD16 and CD94 cell surface molecules. This study reports that NK cells also produce IL-5, a cytokine typically produced by Th2 cells, which mediates mobilization and differentiation of eosinophils. Polyclonal NK cell populations and NK cell clones produce IL-5 when stimulated to proliferate with gamma-irradiated MM-170 melanoma cells or JY B-lymphoblastoid cells and rIL-2. IL-5 is produced in cultures generated from freshly isolated NK cells (primary cultures) and when quiescent NK cells from primary cultures are restimulated to proliferate (secondary cultures). Production of IL-5 is on average 8.8-fold greater in secondary cultures compared with primary cultures (n > 18), suggesting that the ability of NK cells to produce IL-5 matures during primary stimulation. IL-5 secretion, particularly in primary cultures, is augmented by IL-4 and is inhibited by IL-12 and IL-10. By contrast, IL-4 and IL-12 have the reverse effects on IFN-gamma secretion. Cultured NK cells that no longer secrete cytokines can be restimulated to do so with either phorbol 12, 13 dibutyrate and ionomycin or with susceptible target cells in the presence of rIL-2. IL-5 production in these cultures occurs only when NK cells are in an exponential growth phase, whereas IFN-gamma, TNF-alpha, and granulocyte macrophage-CSF are produced also by stimulation of quiescent cells, although to a lesser extent. Furthermore, cytokine production is unrelated to the cytolytic activity of NK cells. In conclusion, proliferating human NK cells have the potential to produce IL-5 with secretion regulated by IL-4, IL-10, and IL-12.

Antibodies, Monoclonal↗

PubMed1995

Distinct granzyme expression in human CD3- CD56+ large granular- and CD3- CD56+ small high density-lymphocytes displaying non-MHC-restricted cytolytic activity.

Cultured natural killer (NK) cells derived from CD3- CD56+ high-density small lymphocytes (HDLs) exhibit similar morphology and high levels of non-major histocompatibility complex-restricted (NK) cytotoxicity equivalent to those of cultured NK cells from CD3- CD56+ low-density large granular lymphocytes (LGLs). To examine the similarities and differences between NK cells from HDLs and NK cells from LGLs, we investigated the expression of three distinct members of the granule serine protease (granzyme) family within cultured CD3- CD56+ LGLs and HDLs. CD3- subpopulations of nonadherent peripheral blood mononuclear cells, LGLs (density < 1.063 g/ml), and HDLs (density > 1.063 g/ml) were stimulated to proliferate in culture. The cultured cells from each population were entirely CD3- CD56+ and were indistinguishable in terms of their increased granularity and size once activated. All cultured CD3- CD56+ LGLs and HDLs displayed cytolytic activity against K562 and immunoglobulin-coated P815. Western analysis detected perforin in both cultured LGL and HDL populations. Cultured HDLs and LGLs both expressed BLT-esterase activity and human granzyme A mRNA. Granzyme B mRNA and protein and Asp-ase activity were detected in unstimulated and cultured LGLs and cultured HDLs. By contrast, unstimulated HDLs did not express significant levels of granzyme B. High levels of Hu-Met-1 granzyme mRNA and Met-ase activity were detected only in cultured LGLs. Thus, despite the development of large granular morphology during proliferation, interleukin-2 cultured CD3- CD56+ HDLs display a different pattern of granzyme expression from CD3- CD56+ LGLs. These data also further suggest an unusually restricted expression of the Hu-Met-1 granzyme in LGLs.

Amino Acid Sequence↗

PubMed1994

A novel granulocyte-derived peptide with lipopolysaccharide-neutralizing activity.

Rabbit CAP18 (cationic antimicrobial protein, 18 kDa) is a leukocyte protein identified and purified using as an assay its capacity to bind and inhibit various activities of LPS. Oligonucleotide probes designed from the putative N-terminal protein sequence were used to obtain the corresponding cDNA from a rabbit bone marrow cDNA library. Examination of the cDNA sequence revealed that the protein fragment of the putative N-terminus was actually a 37-amino-acid C-terminal fragment. This fragment, designated CAP18(106-142), inhibits many activities of LPS. In the present studies, synthetic CAP18(106-142) is shown to: 1) bind to erythrocytes coated with diverse strains of LPS; 2) inhibit LPS-induced release of cytokines (TNF, IL-1, IL-6) and nitric oxide from macrophages; 3) inhibit LPS-induced LAL coagulation and 4) protect mice from LPS lethality. CAP18(106-142) may have therapeutic utility for conditions associated with elevated concentrations of LPS.

Amino Acid Sequence↗

PubMed1994

Molecular associations involving CD16, CD45 and zeta and gamma chains on human natural killer cells.

zeta (CD3-zeta) and gamma (Fc epsilon RI gamma) chains associate with CD16, the low affinity receptor for IgG (Fc gamma RIII) on human NK cells and are essential for the cell surface expression of CD16 and for CD16-mediated effector functions. This study has investigated whether, on NK cells, molecules other than CD16 associate with zeta and gamma chains, as a method of identifying other NK cell surface molecules important in NK cell function. Cell surface biotinylated NK cells were lysed in digitonin, and the lysates immunoprecipitated with mAb to CD16, zeta and gamma, and the immunoprecipitates analysed by SDS-PAGE. CD16 mAb co-precipitated zeta and gamma chains (16 and 12kD, respectively) and in addition molecules of 24, 32-35, 100, 150 and 180-200 kD. Also, zeta mAb co-precipitated gamma chain, and molecules of 24-26, 32-35, 48, 50-66, 100, 150 and 180-200 kD; and gamma co-precipitated zeta chain, and molecules of 24-26, 29, 32-35, 37, 45, 49, 50-66 and 100 kD. While significant amounts of zeta and gamma were co-precipitated with CD16, 10 to 12-fold more zeta and gamma were immunoprecipitated with their respective mAb. Furthermore, depletion of CD16 from the lysate resulted in only a partial (10-12%) depletion of zeta and gamma, indicating that only a relatively small proportion (10-12%) of these molecules are associated with CD16. Interestingly, substantial amounts of molecules with electrophoretic mobility similar to CD16 (50-66 kD) were co-precipitated with zeta and gamma chain mAb from lysates depleted of CD16. In contrast to NK cells where zeta associated with a number of different molecules, the majority of zeta in T cells was found to be associated only with the TCR:CD3 complex. NK cells showed a strong association between CD45, CD16 and a 33 kD molecule and often a strong association of zeta with CD16, CD45 and an unidentified molecule of approximately 150 kD. Our results show first, that CD16, zeta and gamma each can be efficiently labelled by cell surface biotinylation, and second, that CD16, zeta and gamma each can form a complex with each other, and with a number of additional molecules including a 33 kD molecule and CD45 potentially important in NK cell function.

Antibodies, Monoclonal↗

PubMed1994

Effects of the anti-inflammatory compounds castanospermine, mannose-6-phosphate and fucoidan on allograft rejection and elicited peritoneal exudates.

The glycoprotein processing inhibitor castanospermine (CS) and the monosaccharide mannose-6-phosphate (M6P), as well as some sulfated polysaccharides (SPS), have been shown to inhibit inflammation in rat models of experimental autoimmune encephalomyelitis and adjuvant-induced arthritis. Here, the anti-inflammatory effects of these agents have been further explored in murine models of allograft rejection and elicitation of peritoneal exudates. CS, M6P and the SPS, fucoidan, partially inhibited rejection of permanently accepted thyroid allografts induced by the i.p. injection of donor strain (H-2d) spleen cells with a reduction in leucocyte infiltration of 25-36%. However none of these agents reduced the more extensive leucocyte infiltration induced by the i.p. injection of P815 (H-2d) unless recipient mice were pretreated with the immunosuppressant, cyclosporin A (CsA). Elicitation of peritoneal exudates by thioglycollate was inhibited by CS, M6P and fucoidan with sustained leucopenia being induced by CS. In contrast, CS and fucoidan, but not M6P, inhibited antigen-elicited peritoneal exudates. These results suggest that CS, M6P and the SPS fucoidan exhibit subtle differences in their anti-inflammatory activity but probably inhibit inflammation at the level of leucocyte extravasation.

Animals↗

PubMed1994

Differential expression of CD45R0 on natural killer (NK) cells in patients with an NK lymphocytosis.

NK cells from three donors with a NK (CD3- CD56+ CD16+) lymphocytosis of unknown aetiology showed differential expression of CD45R0, an isoform of CD45 not expressed by NK cells from normal donors unless stimulated to proliferate in vitro. For donor FC, 60% of NK cells expressed CD45R0 over a 16 month period during which there was a partial resolution of the NK lymphocytosis. For donor SW, 37% of NK cells expressed CD45R0, increasing to 87% over a 14 month period during which the NK lymphocytosis increased. For donor RN few if any NK cells expressed CD45R0. After in vitro proliferation, 100% of NK cells generated from all donors expressed CD45R0. For donors FC and SW, CD45R0 remained expressed on more than 90% of cells at 3-4 weeks following cessation of proliferation. By contrast CD45R0 expression was gradually lost during long-term culture of NK cells from donor RN, with 58% of NK cells regaining the pre-culture CD45R0- phenotype. NK cells from normal donors also varied in the extent to which activation acquired CD45R0 was lost during long-term culture. The results obtained are consistent with the notion that NK cells from the NK lymphocytosis donors studied have previously undergone proliferation in vivo.

Adult↗

PubMed1994

Cellular distribution of endotoxin after injection of chemically purified lipopolysaccharide differs from that after injection of live bacteria.

Lipopolysaccharide (LPS) chemically extracted from gram-negative bacteria is often used in animal models to study endotoxemia. Laser confocal microscopy and immunofluorescence staining for comparison of injections of live Escherichia coli O111:B4 bacteria with LPS extracted from the same strain showed that cellular localization and time course in rat organs were markedly different after the two injections. Fluorescent staining and image analysis software allowed quantitative comparison of LPS within tissues at different times and doses. Antigenic LPS was detected in all tissues 1 hour after injection of both bacteria and LPS and was present in liver and spleen over the 28-day study period. Whole bacteria were identified in tissue macrophages for the first 48 h after injection; later, bacterial cell walls were replaced by diffuse antigenic material throughout the cytoplasm. Antigenic LPS was localized within hepatocytes only after injection of chemically purified LPS. Cellular localization of LPS in tissues is dependent on the form injected. Animal models that use purified LPS may not be representative of gram-negative bacteremia.

Animals↗

PubMed1994

Synthetic peptides that mimic the binding site of horseshoe crab antilipopolysaccharide factor.

Tachypleus antilipopolysaccharide (LPS) factor (TALF) is a protein of 102 amino acids in the lysate of amebocytes of Tachypleus tridentatus that binds bacterial LPS with high affinity and blocks its biologic activity in numerous assays. To elucidate the minimal sequences that bind LPS, overlapping synthetic peptides based on the sequence of TALF were assessed for the ability to bind and neutralize LPS. TALF41-53 was the minimal sequence that bound LPS, as assessed by a slot blot capture assay. TALF29-59 bound LPS with the highest potency. TALF29-59 decreased LPS-induced coagulation of limulus amebocyte lysate, induction of cytokines from human monocytes, and LPS-induced lethality in sensitized mice. Synthetic peptides based on TALF or other LPS-binding proteins may be useful for the design of drugs for treatment of endotoxemia.

Amino Acid Sequence↗

PubMed1994

Proliferation of alloreactive human natural killer cells independent of specific allogeneic stimulation.

This study establishes that natural killer (NK) cells cytolytic for B-lymphoblastoid cell lines (B-LCL) expressing NK-defined alloantigens can be stimulated to proliferate in culture independently of allogeneic stimulation. NK cells proliferate following co-culture with a gamma-irradiated malignant melanoma cell line (MM-170) and IL-2-conditioned medium. The cultured NK cells from some donors showed a high level of cytotoxicity against NK-1+ B-LCL and this corresponded with a high precursor frequency (46-64%) determined from limiting dilution analysis. Alloreactive NK cells proliferated in cultures containing autologous activated T cells, demonstrating that alloantigens were not essential to stimulate proliferation. B-LCL expressing NK-1 or NK-2 or neither of these alloantigens stimulated proliferation of NK cells cytolytic for NK-1+ B-LCL. Studies using metabolically inactivated B-LCL confirmed that stimulation was not alloantigen dependent. The results demonstrate that recognition of alloantigens by NK cells, sufficient to trigger their lytic program, is not required and indeed is not sufficient to confer a stimulatory signal for proliferation of alloreactive NK cells.

Cells, Cultured↗

PubMed1993

CD48 is a low affinity ligand for human CD2.

COS cells transiently transfected with human CD48 were found to bind human PBL, whereas mock or CD7-transfected COS cells failed to bind human lymphocytes. Binding of PBL to CD48 transfectants was almost totally inhibited by either CD48 mAb pretreatment of COS cells or CD2 mAb pretreatment of PBL, implying an interaction between CD2 and CD48. This conclusion was confirmed by the demonstration that a highly fluorescent, multimeric form of rCD2 bound to CD48 transfected COS cells in a CD48-dependent manner. Additional mAb blocking studies revealed that CD48 interacts with the T11(1) region of CD2, the same region of CD2 that binds LFA-3 (CD58). Thus, CD48 and CD58 represent alternative and possibly competing ligands for CD2, although based on blocking studies with soluble CD2, CD48 interacts with CD2 with approximately a 100-fold lower affinity that CD58.

Antibodies, Monoclonal↗

PubMed1993

Detection of a glycosylation-dependent ligand for the T lymphocyte cell adhesion molecule CD2 using a novel multimeric recombinant CD2-binding assay.

The CD2 molecule plays an important role in T cell adhesion by interacting with the ligands CD58 (LFA-3) and CD59. In order to detect additional ligands for CD2, potentially of low binding affinity, we have prepared a highly fluorescent, multimeric form of rCD2 whose binding to cells can be quantified by flow cytometry. Initial studies demonstrated that binding of multimeric rCD2 to cells was CD2-specific, concentration and time dependent, and saturable. The negative charge on cells was also found to play a critical role in the efficiency of multimeric rCD2 binding. Analysis of binding of multimeric rCD2 to 17 CD58+ cell types revealed that only 8 of the cells exhibited binding. Failure of multimeric rCD2 to interact with the other cells could not be explained by differences in CD58 expression, suggesting that, in terms of CD2 binding, there are qualitative differences in CD58 on different cell types. Binding of multimeric rCD2 to six of the seven reactive cells was virtually totally inhibited by CD58 mAb pretreatment, whereas binding to the erythroleukemic line K562 was only partially blocked, suggesting the existence of another CD2 ligand. Subsequent studies demonstrated that the putative new ligand is not CD59, and that it interacts with a different region of the CD2 molecule than CD58, probably a site located between the T11(1) and T11(2) epitopes. The binding affinity of CD2 for the new ligand is 10-fold lower than for CD58 and, based on studies with truncated rCD2, the binding site for the new ligand is located within the amino-terminal 105 amino acids of the CD2 polypeptide. Unlike CD58, the new ligand is tunicamycin sensitive suggesting that it contains a N-linked carbohydrate structure that is essential for functional activity.

Antibodies, Monoclonal↗

PubMed1993

Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide.

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram-negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti-lipid A mAbs function by neutralizing the toxic effects of LPS.

Adult↗

PubMed1993

T lymphocyte responses to antigens of gram-negative bacteria in pyelonephritis.

We showed previously that large numbers of T lymphocytes accumulate within a few days in the kidneys of rats with ascending pyelonephritis induced with Escherichia coli or Pseudomonas aeruginosa. CD4+ T cells propagated from the lesions exhibited MHC-restricted proliferative responses to formalin-fixed bacteria of the species used to induce infection. In the present study we investigated further the nature of the antigens responsible for the T cell proliferation and studied the ability of different bacterial strains and species to produce proliferative responses. We found that heat-killed bacteria were more stimulatory than formalin-fixed bacteria, and that soluble supernatants of heat-killed organism were also effective. The stimulatory effects of supernatants were destroyed by trypsin and the responses were MHC-restricted. Twelve different E. coli strains, with or without characteristics of uropathogenicity in humans, were all highly stimulatory to T cells derived from a kidney infected with a single E. coli strain. Strains of Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia marcescens--species of Enterobacteriaceae closely related to E. coli--were also stimulatory, whereas more distantly related bacteria--Proteus, Morganella, and P. aeruginosa--were not. T cells propagated from kidneys infected with P. aeruginosa responded to supernatants of this organism, but not to E. coli supernatants. We conclude that a protein antigen (or antigens) shared by strains of E. coli and related Enterobacteriaceae, but not by other gram-negative bacteria, produce MHC-restricted proliferative responses of CD4+ T cells that infiltrate rat kidneys infected with E. coli.

Animals↗

PubMed1993

Human natural killer (NK) cells: requirements for cell proliferation and expansion of phenotypically novel subpopulations.

Previous studies established that the high density (resting) natural killer (NK) cell subset (R-NK) of peripheral blood NK cells is unresponsive to interleukin-2 (IL-2) but can be induced to proliferate when cultured with gamma-irradiated malignant melanoma (MM-170) cells or mitomycin-C-treated activated T cells in the presence of an IL-2-conditioned medium (IL-2-CM). This study has examined additional requirements of this activation process. The induction of proliferation was dependent on cell to cell contact with metabolically active stimulator cells, although no evidence was obtained that stimulation was effected by soluble factors produced by the stimulator cells. Compared with IL-2-CM, rIL-2 was an inefficient costimulator for the induction of NK cell proliferation, suggesting that factors in IL-2-CM were required in addition to IL-2, but rIL-2 was as efficient as IL-2-CM in maintaining the proliferation of activated NK cells. Under optimum culture conditions, NK growth of up to 3200-fold occurred during a proliferation cycle of 18 days. Phenotypic analysis of the culture-generated quiescent NK cells revealed novel heterogeneity in CD16 (Fc gamma RIII) and CD56 (N-CAM) expression. Some NK cells lacked expression of both CD16 and CD56 (as identified using currently available monoclonal antibodies), while other NK cells showed differential CD16 epitope expression. Since quiescent NK cells can be obtained in large numbers and high purity, they will be a convenient source of NK cells to study the molecular processes involved in initiating NK cell proliferation.

Cell Division↗