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Biomedical subjects

C R Lyons

Publications and source records attributed to C R Lyons.

At least 19 recordsLinked to original sources
PubMed1997

Expression of lung inducible nitric oxide synthase protein does not correlate with nitric oxide production in vivo in a pulmonary immune response against Cryptococcus neoformans.

Mice infected intratracheally with Cryptococcus neoformans (Cne) require CD4 and CD8 T cells, IFN-gamma, and M phi production of nitric oxide (NO) for effective resolution of the pulmonary infection. Differences exist among strains of mice in clearing the infection. C.B-17 mice reduced Cne lung burden at a significantly greater rate than C57BL/6 (B6) mice and resistance correlated with greater IFN-gamma production by C.B-17 lung-associated lymph node cells. We examined whether the differences observed in the ability of B6 vs C.B-17 mice to clear Cne was due to 1) numbers of inflammatory cells recruited to the lung, 2) the activation state of the recruited cells as measured by expression of inducible nitric oxide synthase (iNOS), and/or 3) the in vivo production of NO as measured by quantitating urine nitrates. The level of iNOS protein was identical in lungs from both strains of mice during Cne infection as determined by Western blot analysis of whole lung homogenates and immunocytochemistry of isolated lung macrophages. Surprisingly, in vivo studies of iNOS activity indicated that NO production in B6 mice was significantly less than that in C.B-17 mice. While single cell suspensions from lungs of either mouse strain produced identical amounts of NO, NO production by lung explants paralleled in vivo urinary nitrate excretion, suggesting that the maintenance of pulmonary architecture and cell-cell interaction was necessary for suppression of iNOS activity in B6 mice. These data strongly implicate the existence of mechanisms that regulate NO production at the level of enzyme activity during infections and have important implications for analyzing the role of iNOS during an immune response in in vivo models.

Animals↗
PubMed1997

Immunoglobulin response to intrapulmonary immunization of asthmatics.

Atopic asthmatics, compared to non-atopic individuals, exhibit an increased amount of serum antigen-specific IgE and IgG4 antibody directed toward many aeroallergens. We tested the hypothesis that this difference between atopics and non-atopics extends to the response to intrapulmonary deposition of a neoantigen, keyhole limpets haemocyanin (KLH). We immunized nine atopic asthmatics and nine non-atopic controls with 500 micrograms KLH instilled into a subsegment of the lingula and examined serum anti-KLH, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM and specific antibody production by peripheral blood mononuclear cells for 25 days. We also determined specific antibody in bronchoalveolar lavage fluid (BALF) in both the immunized and a non-immunized lobe 11 days after immunization. We found specific serum antibody in all immunized subjects with no difference between atopics and normals in the amount or kinetics of anti-KLH IgG1, IgG2, IgG3, IgA1, IgA2 and IgM. However, the atopics exhibited more anti-KLH IgG4 than the normal controls. Specific anti-KLH antibody-producing cells were detected in peripheral blood in most subjects at day 8 to 12 after immunization with no difference between atopics and normals. Specific IgA1, IgA2, IgG1 and IgM antibodies were detected in BALF from the immunized lobes but not from the non-immunized lobes of both groups of subjects with no difference between atopics and normals. We conclude that atopic asthmatics respond to intrapulmonary KLH with more serum anti-KLH IgG4 than normal controls, consistent with a bias toward a Th2 response to intrapulmonary exposure to antigen.

Adolescent↗
PubMed1996

Emerging roles of nitric oxide in inflammation.

The short-lived, highly reactive NO radical arises in two ways: 1) a rapidly triggered process well suited for homeostatic fine-tuning of blood pressure and 2) a prolonged high-level production best suited for microbicidal activity. Roles in vascular "leakiness" and leukocyte extravasation are also emerging. Continued investigation may yield new therapies addressing individual facets of inflammation.

Animals↗
PubMed1995

The regulation of pulmonary immunity.

No evidence has emerged which suggests that the principles of immunity derived from studies on cells from other body sites are contradicted in the lung and its associated lymphoid tissue. What is clear, however, is that the environment dictates the types of cells, their relationship to one another, and what perturbing events will set in motion either the development of an "active" immune response or tolerance. Investigating mechanisms for the development of lung immunity has increased our understanding of how human diseases develop and is continuing to suggest new ways to manipulate pulmonary immune responses. Demonstration that lung cells regulate both nonspecific inflammation and immunity through the expression of adhesion molecules and the secretion of cytokines offers hope for ways to design more effective vaccines, enhance microbial clearance in immunosuppressed hosts, and to suppress manifestations of immunologically mediated lung disease. Important lung diseases targeted for intensive research efforts in the immediate future are tuberculosis, asthma, and fibrotic lung disease. Perhaps even the common cold might be conquered. Considering the pace of current research on lung immunity, it may not be too ambitious to predict that these diseases may be conquered in the next decade.

Animals↗
PubMed1995

A role for gamma interferon-induced nitric oxide in pulmonary clearance of Cryptococcus neoformans.

Increasing evidence indicates that T cell-dependent, interferon gamma (IFN gamma)-induced activation of murine macrophages and nitric oxide (NO) production plays an important role in host defenses against many microorganisms. A role for this mechanism in pulmonary defenses against infectious agents has not been examined. Previous studies demonstrated that both CD4 and CD8 T cells were required for lung clearance of encapsulated Cryptococcus neoformans (Cne). The current studies investigated whether IFN gamma-induced NO production was involved in the protective T cell-mediated immune response against Cne. Intratracheal inoculation of a low-virulence strain of Cne into mice resulted in an infection that was progressively cleared in immunocompetent C.B-17, but not severe combined immunodeficient (SCID) mice. The onset of Cne lung clearance in immunocompetent mice coincided with a marked increase in inflammatory cells in the lung, local expression of IFN gamma-inducible nitric oxide synthase (iNOS) messenger RNA (mRNA), and an increase in systemic NO production as measured by urinary nitrate excretion. None of these changes were observed in infected SCID mice. Inflammatory lung cells isolated from Cne-infected C.B-17 mice inhibited the growth of endogenous Cne in vitro by a NO-dependent mechanism. Moreover, lung clearance of Cne in immunocompetent mice was blocked by treatment with (1) antibody to IFN gamma, which blocked iNOS gene expression and NO production, or (2) the arginine analogue, NGmonomethyl-L-arginine (MMA), which only blocked NO production. However, neither anti-IFN gamma nor MMA treatment decreased the numbers or types of recruited inflammatory cells. Thus, these studies demonstrated that, although recruitment of effector cells was required, it was not sufficient to initiate clearance of Cne from the lung. Rather, an IFN gamma-induced effector mechanism, i.e., NO production, was also required.

Animals↗
PubMed1993

Characterization of the third member of the MCAT family of cationic amino acid transporters. Identification of a domain that determines the transport properties of the MCAT proteins.

We have identified the third member of a family of cationic amino acid transporters in lipopolysaccharide-stimulated murine macrophages. The deduced amino acid sequence of this transporter is the same as MCAT-2 (mouse cationic amino acid transporter-2), the low affinity transporter expressed in hepatocytes, except for a stretch of 41 amino acids that connects the eighth and ninth membrane-spanning domains. These transporters apparently result from differential splicing of transcripts from a single gene and therefore have been named MCAT-2A (hepatocyte) and MCAT-2B (macrophage). Despite their similarity, MCAT-2B is saturated at one-fifth the arginine concentration, has a lower apparent Vmax, and is more sensitive to trans-stimulation than MCAT-2. Introduction of the unique regions of MCAT-2A and MCAT-2B into the equivalent portion of the related protein, MCAT-1, created chimeric transporters with properties most like the donor of this region. Our findings suggest these 41 amino acids contain a domain that binds the amino acid substrate during its translocation across the membrane.

Amino Acid Sequence↗
PubMed1993

The role of T lymphocytes in pulmonary microbial defense mechanisms.

Understanding how lung immunity develops against pulmonary pathogens should lead to more rational approaches in vaccine design and to the use of recombinant cytokines in lung disease. T lymphocytes are central to the development of effective immune responses; therefore, understanding how lung immunity develops will require a study of how and where T cells respond to respiratory antigens. Our laboratory has helped define the phenotype and function of lung dendritic cells, which likely play an essential role in stimulating naive T cells to respond to antigens. We found that both interstitial and alveolar macrophages can regulate the function of these cells, the former to enhance activity, the latter to suppress. In addition, we developed a murine pulmonary infection model using the fungus, Cryptococcus neoformans, in which T-cell-mediated immunity is essential for effective host clearance of the organism. The role of T cells in this model is to recruit and activate effector cells to resolve the lung infection; both CD4 and CD8 T-cell subsets are required for optimal effector cell recruitment. These studies are summarized as examples of current approaches to understanding pulmonary immunity.

Animals↗
PubMed1992

Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line.

Macrophages activated by exposure to cytokines and/or to endotoxin produce nitric oxide (NO.), a free radical that is a mediator of the host response to infection. Activation induces the expression of nitric oxide synthase, the enzyme that catalyzes formation of NO. from L-arginine and molecular oxygen. We report the cloning of a cDNA encoding the inducible nitric oxide synthase from a murine macrophage cell line, RAW264.7, exposed to interferon-gamma and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO.. Nitric production is blocked by the enzyme inhibitor, NG-monomethylarginine, and is independent of calcium/calmodulin. RAW264.7 cells demonstrate rapid accumulation of the nitric oxide synthase-encoding mRNAs upon activation. Comparison of the deduced amino acid sequence to the calcium/calmodulin-dependent nitric oxide synthase previously purified (Bredt, D. S., and Synder, S.H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685) and cloned (Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Synder, S. H. (1991) nature 351, 714-718) from rat brain identifies shared binding sites for the cofactors NADPH and flavins in the C-terminal half of both proteins and an additional conserved region near the N terminus that may recognize L-arginine and/or contribute to the active site.

Amino Acid Oxidoreductases↗
PubMed1987

Characteristics of partially purified nerve growth factor receptor.

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Gland Neoplasms↗
PubMed1987

Mononuclear cells in human lung parenchyma. Characterization of a potent accessory cell not obtained by bronchoalveolar lavage.

Human alveolar macrophages from bronchoalveolar lavage are usually poor accessory cells for antigen-induced T-lymphocyte proliferation and poor stimulators of allogeneic mixed leukocyte reactions (MLR) when compared to peripheral blood monocytes. In contrast, cells harvested from minced lungs are good stimulators of a MLR. We have characterized the accessory cells obtained after enzymatic digestion of human lung tissue. Pulmonary mononuclear cells were separated from the dissociated lung cell mixture on Ficoll-Hypaque. Loosely adherent cells (LAM) were obtained after an overnight incubation on plastic culture dishes of initially adherent mononuclear cells. LAM were significantly more effective than were pulmonary mononuclear cells (p less than 0.05), firmly adherent cells (p less than 0.05), alveolar macrophages obtained by bronchoalveolar lavage (p less than 0.05), or monocytes (p less than 0.05) in stimulating allogeneic resting T-cells. Addition of indomethacin and catalase markedly improved T-cell proliferation induced by LAM. Enrichment for Fc receptor negative or for nonphagocytic cells further enhanced the MLR-stimulating capacity of LAM. Phase-contrast studies demonstrated an enrichment in cells compatible with dendritic cells in LAM as compared to firmly adherent cells. We conclude that there are potent accessory cells in human lung that are loosely adherent, Fc receptor negative, and poorly phagocytic, and thus are dissimilar from classic macrophages. We hypothesize that cells similar to dendritic cells might play a role in the initiation of immune responses in lung parenchyma.

Antibodies, Monoclonal↗
PubMed1986

Inability of human alveolar macrophages to stimulate resting T cells correlates with decreased antigen-specific T cell-macrophage binding.

Alveolar macrophages (AM) from the majority of human volunteers are defective antigen presenting cells (APC) in T cell proliferation assays despite the display by the cells of HLA-D region antigens. We have confirmed that AM secrete relatively little interleukin 1 (IL 1), but addition of exogenous IL 1 did not improve the capacity of AM to initiate antigen-induced T cell proliferation. Thus, the presence of HLA-D region antigens and IL 1 is not sufficient to enable an accessory cell to act as an APC. We developed a T cell-accessory cell binding assay to investigate early events in T cell activation. AM demonstrated a diminished capacity as compared with monocytes to bind antigen-specific T cell clones. Nevertheless, AM often induced proliferation of T cell clones as effectively as monocytes, indicating that antigen display was intact. The inefficiency of AM in bind T cell clones correlated with their reduced capacity to induce resting T cells to express IL 2 receptors, secrete IL 2, and proliferate in response to antigen. Indirect immunofluorescence established that similar percentages of AM and monocytes expressed LFA molecules, but the density of the molecules was greater on monocytes than AM. A role for LFA antigens in the physical binding of T cells to monocytes was demonstrated by blocking antigen-specific binding with a monoclonal antibody to LFA-1 antigen. LFA-1 antibody also blocked the low levels of specific binding between AM and T cell clones, indicating that LFA-1-ligand interactions were operative between these two cell types. These studies indicate that there are critical cell membrane characteristics that promote binding of T cells to APC in addition to T cell receptor-antigen interactions. This combination of nonspecific and specific interactions leads to avid T cell-APC binding that may be essential for activation of resting T cells. Furthermore, we postulate that the failure to AM to act as effective APC results from an inability to bind T cells efficiently.

Adult↗
PubMed1986

Human pulmonary macrophages. Functional comparison of cells obtained from whole lung and by bronchoalveolar lavage.

Previous studies have demonstrated that pulmonary macrophages (PM) recovered from bronchoalveolar lavage (BAL) are relatively poor accessory cells for antigen-induced T-lymphocyte proliferation. These studies have suggested that the immune function of macrophages obtained by BAL is representative of the majority of PM. We compared macrophages obtained by BAL with PM from whole lung minces (MIN) for their ability to stimulate T-lymphocyte proliferation. Both populations of PM had similar expression of HLA-DR antigen and were of comparable maturity as determined by staining for MO2 antigen. Production of interleukin 1 by both groups of PM was similar and was significantly less than that produced by monocytes (p less than 0.05). Both populations of PM functioned poorly as antigen-presenting cells when compared with monocytes (p less than 0.05). However, PM from MIN stimulated a mixed leukocyte reaction significantly more (p less than 0.05) than did PM from BAL. Our data suggest that whereas BAL obtains a population of PM with an immunologic function that is largely similar to PM obtained from whole lung, some differences in function may exist.

Antigen-Presenting Cells↗
PubMed1986

Human alveolar macrophages: HLA-DR-positive macrophages that are poor stimulators of a primary mixed leukocyte reaction.

Previous studies demonstrated that alveolar macrophages (AM) from most normal human volunteers failed to stimulate the antigen-induced proliferation of peripheral blood T lymphocytes although greater than 90% of AM expressed HLA-DR antigens. The current studies establish that AM also fail to induce allogeneic peripheral blood mononuclear cells to proliferate in a mixed leukocyte reaction (MLR). Suppressive activity by AM was not an explanation for their failure to induce an MLR. Indirect immunofluorescence established the presence of both HLA-DR and DQ antigens on the majority of AM and the persistence of these antigens on cells in culture for up to 6 days, the period of time required to observe a maximal MLR. Metabolic labeling experiments also demonstrated that HLA-DR antigens were synthesized by AM. It was recently reported that AM secrete relatively small amounts of IL 1, an important ancillary signal provided by accessory cells to enhance the stimulation of lymphocyte proliferation. However, addition of optimal concentrations of IL 1 to cultures containing AM failed to enhance the MLR. Thus, there is at least one additional, but as yet undefined, requirement for an accessory cell to induce an optimal MLR besides the display of HLA-D region antigens and the secretion of IL 1. In contrast, AM were effective in specifically stimulating proliferation of alloreactive T cell lines, suggesting that at least some cell lines do not require this nonspecific undefined second signal. We speculate that although AM may not initiate primary immune responses in the lung, they may be important in maintaining immune-mediated inflammatory responses by specifically restimulating already activated T cells.

Adult↗
PubMed1983

Binding constants of isolated NGF-receptors from different species.

It is known that NGF-responsive cells bind NGF at cell surface receptors in a specific and saturable fashion and there are two separate kinds of receptor-ligand binding interactions as judged by Rosenthal analyses. Following isolation of nerve growth factor receptors from embryonic chicken sensory ganglia, rat pheochromocytoma cells and human neuroblastoma cells, equilibrium binding studies were carried out and two different equilibrium binding constants similar to that described for whole cells were determined. This evidence is consistent with the hypothesis that there are two different receptors for NGF which have been conserved.

Adrenal Gland Neoplasms↗
PubMed1983

Persistence of influenza as an immunogen in pulmonary antigen-presenting cells.

Influenza antigens inoculated into the lung induce local immune responses. It has been proposed that this induction might be partly regulated by local antigen-presenting cells. The purpose of the current study was to inoculate heat-inactivated influenza virus into the tracheae of guinea pigs and determine the quantity of antigens that became cell-associated. Second, we determined how long antigen-presenting bronchoalveolar cells that had taken up virus in vivo retained their ability to specifically stimulate virus-immune T lymphocytes. Radioiodinated heat-inactivated influenza virus was inoculated into the tracheae of guinea pigs. The animals were killed from 30 min to 14 days after intratracheal inoculation, and radioactivity was determined in cells isolated from lung tissue. At least one-third of the radioactivity in the lungs was cell-associated from 1 to 14 days post-inoculation. In separate studies, heat-inactivated virus was inoculated into the airways of guinea pigs, and animals were killed at various times thereafter. Bronchoalveolar cells from these animals were compared with those from uninoculated controls in their ability to specifically stimulate virus-immune T cells to proliferate in vitro. Bronchoalveolar cells from virus-inoculated animals specifically stimulated T lymphocytes for up to 7 days after virus inoculation. These studies suggest that immunogenic virus persists in the lung within antigen-presenting cells for at least 1 week and possibly for up to 2 weeks. The persisting immunogenic stimulus after the termination of viral infections might be critical in ensuring the development of a local protective immune response.

Animals↗
PubMed1983

Alveolar macrophages in pulmonary immune responses. I. Role in the initiation of primary immune responses and in the selective recruitment of T lymphocytes to the lung.

Antigen inoculated intratracheally (IT) into animals can induce primary immune responses and selectively recruit specific T cells to the lung. In the current study, the role of alveolar macrophages (AM) in these two responses was investigated. Antigen-pulsed bronchoalveolar cells (BAC) inoculated IT into guinea pigs generated a population of immune T cells that proliferated in vitro on reexposure to antigen-pulsed macrophages (Mø). The possibility that antigen-pulsed donor BAC shed antigen that was subsequently processed and presented by host Mø was ruled out by genetic experiments. Thus, peritoneal exudate lymphocytes (PEL) from (2 X 13)F1 guinea pigs primed with antigen-pulsed BAC from strain 2 animals responded preferentially to antigen-pulsed strain 2 Mø rather than to antigen-pulsed strain 13 Mø. In a second set of studies, antigen-pulsed BAC inoculated IT into guinea pigs selectively recruited antigen-specific T cells to the lung. Genetic experiments verified that inoculated BAC were the source of the antigen-presenting cells responsible for selective recruitment. Thus, antigen-pulsed strain 2 BAC inoculated IT recruited a greater proportion of (2 X 13)F1 T cells that recognized antigen in the context of strain 2 Mø than F1 T cells that recognized antigen on strain 13 Mø. Taken together, these studies suggest that AM contribute to the regulation of pulmonary immunity by both inducing T lymphocyte immunity and selectively recruiting specific T cells to the lung.

Animals↗
PubMed1982

The antigen-induced selective recruitment of specific T lymphocytes to the lung.

The purpose of the present studies was to investigate the mechanisms by which specific T lymphocytes accumulate in the lung. After the intratracheal (IT) inoculation of influenza virus into guinea pigs, the detection of specific T lymphocytes in the lung coincided with the development of immunity in both hilar nodes and systemic lymphoid tissue. Animals immunized in the footpads with virus failed to develop immune responses in the lung unless rechallenged IT with immunogen. In adoptive transfer experiments, IT inoculation of influenza into nonimmune guinea pigs, followed immediately by the i.v. injection of a mixture of 3H-thymidine-labeled syngeneic T lymphocytes specific for influenza virus and 14C-thymidine-labeled syngeneic T lymphocytes specific for an irrelevant antigen resulted in the selective accumulation of the virus-specific T lymphocytes in the lung. Taken together, these studies indicate that the selective recruitment by antigen of circulating immune cells is one of the mechanisms by which specific T cells accumulate in the lung.

Animals↗