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Biomedical subjects

T W Klein

Publications and source records attributed to T W Klein.

At least 55 records · Page 3Linked to original sources
PubMed1995

Expression of cannabinoid receptor mRNA in murine and human leukocytes.

delta 9-tetrahydrocannabinol, the major psychoactive cannabinoid of marijuana modulates immune cells in vivo and in vitro. It is possible that the drug exerts it's effect either by inserting into and disrupting the cell membrane (nonreceptor mechanism) or by binding to a cannabinoid receptor moiety and thus altering cell function through some form of signal transduction. In the present study, we confirm and extend the findings that mouse and human immune cells express specific cannabinoid binding sites and cannabinoid receptor mRNA. Reverse transcription-polymerase chain reaction analysis showed the presence of receptor mRNA not only in the neuroblastoma cell line (N18TG-2), but also in mouse splenocytes and in cell lines such as NKB61A2 (a mouse natural killer-like), CTLL2 (a mouse IL2-dependent T cell), THP-1 (a human monocytic cell) and Raji (a human B cell) but not in Jurkat (a human T cell). Furthermore, the receptor mRNA was expressed in purified populations of resting splenic T and B lymphocytes but not in resting populations of enriched splenic macrophages. Finally, LPS-stimulated Raji and PMA-stimulated THP-1 human cell lines showed increased levels of the cannabinoid receptor mRNA. These results suggest cannabinoid receptors have biological relevance in lymphoid cells because: receptor mRNA is detected in some resting immune cells but not others and the mRNA increases during cell activation. The major psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC), has been shown to modulate human and mouse immune responses both in vitro and in vivo (1,2).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Marijuana, receptors and immunomodulation.

THC, the major psychoactive component of marijuana, has been shown both in humans and experimental animals to have immunomodulatory properties. For example, marijuana smokers may show impaired immunological functions, including deficiency of blood leukocyte blastogenesis to mitogens. Detailed studies with mice have shown that animals given THC can show marked immunomodulation, including suppression of antibody formation, deficient cytokine production, etc. However, recent studies have also shown that lymphoid cells evince enhanced production or release or IL1, but suppression of IL2 and interferon production. Such lymphoid cells treated in vitro with THC also show suppressed blastogenesis to antigens and mitogens, suppressed NK activity, etc. In contrast, it has recently been shown that THC can enhance production or release of pro-inflammatory cytokines. This includes release of these cytokines from macrophages, including augmented release of IL1, TNF alpha, and IL6 activity. Susceptibility of mice to infection with opportunistic organisms such as L. pneumophila has been found and this increased susceptibility can be modulated by THC. A toxic shock-like death to Legionella has been induced by THC treatment given one day before and one day after infection. Receptors to THC have been detected in the brain as well as in peripheral tissues, including lymphoid cells. Thus, immunomodulation induced by THC may be related to receptor effects as well as unrelated to such receptors. It is clear that THC and other cannabinoids are excellent tools for studying the mechanisms of immune modulation, especially altered susceptibility to microbial infection.

Adjuvants, Immunologic↗
PubMed1995

IL-1 beta and TNF alpha modulate delta 9-tetrahydrocannabinol-induced catalepsy in mice.

The role of the proinflammatory cytokines interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) in THC-induced catalepsy in mice was examined. Recombinant IL-1 beta (400 ng/mouse, IV) and TNF alpha (500 ng/mouse, IV) were effective in potentiating the cataleptic effect of low-dose THC (10 micrograms/mouse, IV). Recombinant IL-1 alpha and IL-6 did not potentiate catalepsy at any dose tested. Anti-IL-1 beta and anti-TNF alpha antibodies were effective in attenuating high-dose (75 micrograms/mouse) THC-induced catalepsy. Antibodies to IL-1 alpha and IL-6 had no effect on catalepsy. Early onset catalepsy (10 min postinjection) was potentiated by exogenous recombinant IL-1 beta and TNF alpha but only later catalepsy (2 h postinjection) was attenuated by antibodies to endogenous IL-1 beta or TNF alpha. This divergence of the cytokine effect suggests that these substances regulate, by different mechanisms, the early and late THC-induced cataleptic response.

Animals↗
PubMed1995

Quantitative reverse transcription-PCR analysis of Legionella pneumophila-induced cytokine mRNA in different macrophage populations by high-performance liquid chromatography.

Cytokine production in macrophages infected by bacteria is critical for the course of infection. However, it is not known how infection of macrophages with opportunistic bacteria leads to cytokine production in different populations of cells. Since it is possible that cytokine genes may be differentially regulated by attachment rather than by active infection, the levels of various cytokine mRNAs were measured in alveolar macrophages (AMs), peritoneal resident macrophages (RMs), and peritoneally elicited macrophages (EMs) interacting with Legionella pneumophila by using cytochalasin D-treated macrophages and a newly developed quantitative reverse transcription-PCR procedure with high-performance liquid chromatographic analysis to determine cytokine mRNA formation. Increased levels of interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and macrophage inflammatory protein 2 mRNAs were quantitated in the macrophages responding to L. pneumophila attachment in vitro. Using this technique, we showed that the three different macrophage populations responded differently to bacterial attachment. We found that the levels of IL-6 and granulocyte-macrophage colony-stimulating factor mRNAs induced by the attachment of L. pneumophila to AMs were significantly lower than the levels in RMs but similar to the levels in EMs. Furthermore, the levels of MIP-2 mRNA in the AMs were found to be higher than those in the RMs, but similar levels were found in EMs. IL-1 beta mRNA levels were higher in both AMs and RMs than in EMs, but tumor necrosis factor alpha levels were not different among the three macrophage populations examined. Thus, the responses of macrophages to bacterial attachment in terms of cytokine mRNA levels were readily quantitated by the reverse transcription-PCR assay. However, the results obtained showed different levels of responsiveness of distinct macrophage populations to L. pneumophila attachment, and this could be related to the characteristic nature of the macrophage type examined.

Animals↗
PubMed1995

delta 9-Tetrahydrocannabinol, cytokines, and immunity to Legionella pneumophila.

The major psychoactive component of marijuana, delta 9-tetrahydrocannabinol (THC), has been shown to suppress the functions of various immune cells. However, the relationship of these findings to THC-induced suppression of host resistance to infection has not been firmly established. In this report, we review the literature concerning THC's effects on cytokine production and resistance to infection with Legionella pneumophila (Lp). Recent reports have linked THC-induced immunomodulation with drug-induced modulation of the cytokine network. Specifically, THC in vivo suppresses interferon (IFN) production while in vitro modulates the production of tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-2 (IL-2), and interleukin-2 receptor (IL-2R). These results suggested that THC treatment might alter host immunity by disrupting the cytokine network. Immunity and resistance to infection with Lp depends upon the activation of killer cells and the stimulation of the cytokine network. THC injection into rodents was observed to augment acute phase cytokine mobilization in response to a primary Lp infection; on the other hand, the drug suppressed the development of protective immunity and resistance to secondary Lp infection by causing a change in the profile of T helper cell cytokines produced by Th1 and Th2 cells. Thus, it appears that THC injection suppresses resistance to Lp infection by disrupting the cytokine network. Regarding the molecular mechanisms of these effects of THC, data is reviewed concerning the role of cannabinoid receptors (CR) in cells of the immune system. In summary, the literature to date supports the role of THC as an immunomodulator capable of suppressing resistance to infection through mechanisms involving alteration of the cytokine network. The role of CR receptors in these events has yet to be determined.

Cytokines↗
PubMed1995

Legionella pneumophila-induced visual learning impairment reversed by anti-interleukin-1 beta.

Infecting mice with the opportunistic intracellular pathogen Legionella pneumophila markedly inhibited place learning of infected C57BL/6 mice as determined by the Morris water maze test. Mice infected with L. pneumophila evinced much less ability to learn the position of a hidden platform than did normal noninfected mice, which quickly learned the location of the hidden platform and escaped from the cool water of the pool with increasing efficiency. However, infected mice treated with anti-interleukin-1 (anti-IL-1) neutralizing antibody learned the task with about the same efficiency as the controls. When the animals were tested 1 week after learning, control animals remembered the task well and were able to escape with near maximal efficacy. On the other hand, L. pneumophila-infected mice performed as poorly after the 1 week rest as during the training period, indicating that infection blocked learning and not merely performance. Mice infected with L. pneumophila and given the antibody treatment were found to be indistinguishable from controls in that they remembered the task and escaped with good efficiency. Thus, the results of this study suggest that the pro-inflammatory cytokine, IL-1 beta, is involved, at least partly, in the attenuation of spatial navigational learning in mice infected acutely with a sublethal concentration of L. pneumophila. These results, therefore, suggest that cognitive impairment of L. pneumophila-infected mice may be related to the cytokine IL-1 beta and, furthermore, that cytokines may be related to learning and memory changes experienced by individuals suffering acute bacterial infections.

Animals↗
PubMed1995

delta 9-Tetrahydrocannabinol (THC) causes the variable expression of IL2 receptor subunits.

Previously, we reported that the cannabinoid delta 9-tetrahydrocannabinol (THC) suppressed interleukin 2 (IL2)-induced proliferation of a cloned, natural killer-like cell line (NKB61A2) and decreased the number of high- and intermediate-affinity IL2 binding sites. However, the surface expression of interleukin 2 receptor alpha (IL2R alpha) chain, as measured by flow cytometry, was increased rather than decreased by THC treatment. This suggested that the drug-induced deficiency in IL2 binding and cell activation involved a defect in the cell-surface expression of IL2R subunits other than the alpha chain. Because the IL2 receptor complex is composed of alpha, beta and gamma chains, we examined the effect of THC treatment on the expression of these chains. In a result consistent with our previous findings, we observed that treatment of NKB61A2 cells with THC increased the cellular immunoprecipitable IL2R alpha protein (p55) and mRNA. Furthermore, the cellular production of IL2R beta chain protein (p75) and mRNA, determined by immunoprecipitation and Northern blotting, respectively, was also increased. The mRNA stability assay showed that THC increased the stability of IL2 beta mRNA, and nuclear run-on experiments suggested that the increase in subunit production was not due to a drug effect on gene transcription. The IL2R gamma chain was also affected by THC treatment in that Northern blotting studies showed a drug-induced decrease in the cellular level of gamma chain mRNA. In addition, THC treatment decreased the 125I-labeled IL2 internalization under high-affinity binding conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

delta 9-Tetrahydrocannabinol (THC) modulates IL-1 bioactivity in human monocyte/macrophage cell lines.

We have previously observed that delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, increased supernatant interleukin-1 (IL-1) bioactivity in cultures of mouse resident peritoneal macrophages stimulated with lipopolysaccharide (LPS). In this study, experiments were performed to determine whether THC treatment similarly affected phagocytes of human origin. The results showed that THC increased the levels of supernatant IL-1 bioactivity of two human monocytic cell lines, but only if the cells were differentiated with phorbol myristate acetate. Undifferentiated cells displayed decreased IL-1 bioactivity in response to THC. However, under conditions in which THC augmented supernatant IL-1 bioactivity from THP-1 cells, ELISA studies showed that the levels of IL-1 alpha and IL-1 beta were unchanged and decreased, respectively. Furthermore, supernatant interleukin-6 (IL-6) levels were decreased, but tumor necrosis factor (TNF-alpha) levels were increased by THC treatment. These results show that THC treatment modulates cytokine production and/or release by mouse and human macrophages and the drug effects on IL-1-like bioactivity in the supernatants of the human THP-1 cells are due to increased levels of other cytokines, such as TNF-alpha, rather than IL-1 itself.

Animals↗
PubMed1994

Differences and similarities in permissive A/J versus non-permissive BALB/c murine macrophages infected with Legionella pneumophila: the role of iron.

Legionella pneumophila, a facultative intracellular pathogen, replicates within and kills thioglycolate-elicited (TG) macrophages from A/J mice, while growth is inhibited in TG macrophages from BALB/c mice which show no impaired viability. The role of iron in BALB/c and A/J macrophages regarding their permissiveness to L. pneumophila intracellular growth was investigated. We previously reported that TG macrophages from the A/J mouse strain readily supported the intracellular growth of L. pneumophila, while resident macrophages from the same strain of mice were not permissive. Recently we also found that such a difference in permissiveness between both A/J macrophage populations may be explained, at least in part, to intracellular availability of iron. In this report, differences in permissiveness to L. pneumophila growth between A/J TG macrophages and BALB/c TG macrophages was not due to intracellular iron availability. BALB/c and A/J TG macrophages exhibited similar expression of transferrin receptor and cellular iron content. The treatment of BALB/c TG macrophages with different iron compounds, namely ferric nitrilotriacetate (12.5-100 microM), ferric citrate (12.5-100 microM) and transferrin (0.5-5 mg ml-1), did not stimulate the intracellular proliferation of L. pneumophila. The reduction of intracellular iron availability by treatment with antibodies against transferrin receptor or with desferrioxamine suppressed the growth of L. pneumophila within BALB/c TG macrophages, suggesting that these cells do not restrict L. pneumophila growth because of iron. The production of nitric oxide was also similar in both macrophage populations, as measured by the Griess reaction. However, the synthesis of oxygen reactive species was three times higher in non-permissive BALB/c macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Bacterial heat shock proteins directly induce cytokine mRNA and interleukin-1 secretion in macrophage cultures.

Bacterial heat shock proteins (hsp) have been shown to be important immunogens stimulating both T cells and B cells. However, little is known concerning the direct interactions between hsp and macrophages. In this study, we demonstrated that treatment of macrophage cultures with purified bacterial hsp, including Legionella pneumophila hsp60, Escherichia coli GroEL, Mycobacterium tuberculosis hsp70, Mycobacterium leprae hsp65, and Mycobacterium bovis BCG hsp65, increased the steady-state levels of cytokine mRNA for interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor as well as supernatant IL-1 secretion. This effect was shown not to be due to contamination of the hsp preparations with bacterial lipopolysaccharide. However, not all hsp induced cytokines; M. tuberculosis hsp10 showed minimal activity in our study. These results suggest that bacterial hsp might modulate immunity by rapidly and directly increasing cytokine production in macrophages.

Animals↗
PubMed1994

Macrophage permissiveness for Legionella pneumophila growth modulated by iron.

We have investigated the modulation of iron in two populations of macrophages which differ in susceptibility to Legionella pneumophila intracellular proliferation. Previously, we reported that thioglycolate-elicited peritoneal macrophages obtained from the inbred A/J mouse strain readily support the intracellular growth of L. pneumophila, while resident macrophages from the same strain do not. In this study, we show that A/J elicited macrophages exhibit markedly higher expression of transferrin receptor and intracellular iron content than A/J resident macrophages. Furthermore, apotransferrin and desferrioxamine inhibited the intracellular proliferation of L. pneumophila in elicited macrophages, and this suppression was reversed by the additions of Fe-transferrin or ferric nitrilotriacetate. Fe-transferrin and ferric nitrilotriacetate did not further increase the intracellular proliferation of L. pneumophila in thioglycolate-elicited macrophages. However, ferric citrate and ferric nitrilotriacetate stimulated in a dose-dependent manner the growth of L. pneumophila in resident macrophages. Furthermore, equimolar concentrations of desferrioxamine reversed the stimulatory effect of iron in these resident cells. These data provide evidence supporting the hypothesis that differences in susceptibility to L. pneumophila growth between permissive elicited macrophages and nonpermissive resident macrophages from the A/J mouse strain are due to intracellular availability of iron.

Animals↗
PubMed1994

Legionella pneumophila growth restriction and cytokine production by murine macrophages activated by a novel Pseudomonas lipid A.

Peritoneal exudate macrophages from A/J mice activated by purified lipid A preparations from Pseudomonas vesicularis, which contain 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester as the lipid A backbone, restricted the growth of Legionella pneumophila, an intracellular opportunistic bacteria which readily grows in otherwise permissive macrophages from susceptible A/J mice and induced production of the proinflammatory cytokines interleukin 1 and tumor necrosis factor alpha. Activation of the macrophages was similar to that which occurred after stimulation with more conventional lipid A from other bacteria such as salmonellae. A purified fraction A3 preparation from the Pseudomonas lipid A, which lacked only 1 mol of amide-linked fatty acid, in comparison with another fraction (A2), which contained the fatty acid, also markedly activated the usually permissive macrophages from susceptible A/J mice to resist growth of the legionellae. The fraction A3 also induced both interleukin and tumor necrosis factor alpha. These results show that this novel lipid A from P. vesicularis can activate macrophages to resist infection with an opportunistic bacterium in a manner similar to that induced by conventional enterobacterial lipid A and that the hydrophobic portion of this Pseudomonas molecule may have an important role in activation of macrophages.

Animals↗
PubMed1994

Inhibition of Legionella pneumophila growth by gamma interferon in permissive A/J mouse macrophages: role of reactive oxygen species, nitric oxide, tryptophan, and iron(III).

A/J mouse macrophages infected with Legionella pneumophila and treated with gamma interferon (IFN-gamma) in vitro developed potent antimicrobial activity. This antilegionella activity was independent of the macrophage capacity to generate reactive oxygen intermediates, since the oxygen radical scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on the antilegionella activity of IFN-gamma-activated macrophages. Likewise, whereas the ability of IFN-gamma-activated macrophages to synthesize reactive nitrogen intermediates was markedly inhibited by the L-arginine (Arg) analogs, NG-monomethyl-L-arginine and L-aminoguanidine, as well as by incubation in L-Arg-free medium, their ability to inhibit the intracellular growth of L. pneumophila remained intact. The intracellular growth of L. pneumophila in A/J macrophages was inhibited by the iron(III) chelator desferrioxamine and reversed by Fe-transferrin as well as by ferric salts. Additionally, IFN-gamma-activated macrophages incorporated 28% less 59Fe(III) compared with nonactivated cells. Nonetheless, only partial blocking of growth restriction was observed when IFN-gamma-stimulated macrophages were saturated with iron(III). Indole-propionic acid, which appears to inhibit the biosynthesis of L-tryptophan (L-Trp), was an L-Trp-reversible growth inhibitor of L. pneumophila in macrophages, implying that the intracellular replication of this pathogen is also L-Trp dependent. However, an excess of exogenous L-Trp did not reverse the growth inhibition due to IFN-gamma, though a small synergistic effect was observed when the culture medium was supplemented with both iron(III) and L-Trp. We conclude that IFN-gamma-activated macrophages inhibit the intracellular proliferation of L. pneumophila by reactive oxygen intermediate- and reactive nitrogen intermediate-independent mechanisms and just partially by nutritionally dependent mechanisms. We also suggest that additional mechanisms, still unclear, may be involved, since complete reversion was never obtained and since at higher concentrations of IFN-gamma, iron(III) did not induce any significant reversion in the L. pneumophila growth inhibition.

Animals↗
PubMed1994

Binding of Legionella pneumophila to macrophages increases cellular cytokine mRNA.

Infection of macrophages with Legionella pneumophila induces formation of interleukin 1 beta (IL-1 beta), but the molecular basis of this is not understood. Binding of bacteria to macrophage surfaces is the first step in an infection process. Therefore, we examined whether this step was sufficient to increase the cellular level of mRNAs for IL-1 beta and other cytokines. To assess the effect of binding of L. pneumophila on the steady-state levels of cytokine mRNAs, cultures of thioglycolate-elicited macrophages from L. pneumophila-susceptible A/J mice were treated with cytochalasin D and infected with L. pneumophila and the total RNA was extracted for analysis by reverse transcription-PCR with primers for IL-1 alpha, IL-1 beta, IL-6, tumor necrosis factor alpha, granulocyte macrophage colony-stimulating factor, and beta interferon (IFN-beta). L. pneumophila treatment increased the cellular steady-state mRNA levels of all cytokines except IFN-beta. To determine the specificity of this effect, macrophage cultures were treated with cytochalasin D and either bacterial lipopolysaccharide, bovine serum albumin-sensitized latex, Salmonella typhimurium, or Escherichia coli. Lipopolysaccharide treatment increased all mRNAs, bovine serum albumin-sensitized latex had no significant effect, and treatment with S. typhimurium or E. coli increased all mRNAs except that of IFN-beta. These results suggested that the binding of gram-negative bacteria to the macrophage surface was sufficient to induce a unique pattern of cytokine mRNAs. Additional studies that examined the characteristics of the bacterial ligands involved indicated involvement of both heat-labile and heat-stable surface ligands.

Animals↗
PubMed1994

Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by delta-9-tetrahydrocannabinol injection.

Resistance to infection with Legionella pneumophila is primarily dependent upon cell-mediated immunity rather than humoral immunity. Recent evidence suggests that activation of cell-mediated immunity depends on Th1 cells and activation of humoral immunity depends on Th2 cells. In this report, delta 9-tetrahydrocannabinol (THC), the major psychoactive cannabinoid of marijuana and an immunomodulator, suppressed development of secondary immunity to L. pneumophila, which correlated with a reduction in Th1 activity. BALB/c mice, infected with a primary sublethal dose of L. pneumophila, developed resistance to a larger challenge infection 3 to 4 weeks later. However, intravenous injection of THC (4 mg/kg of body weight) 1 day prior to primary infection resulted in increased mortality after the challenge infection. The level of anti-L. pneumophila antibodies in serum increased in both THC-treated and control mice; however, in the THC group IgG1 antibodies which are stimulated by Th2 cells were elevated while Th1-regulated, IgG2a antibodies were depressed. Furthermore, cultured splenocytes from THC-treated mice had less L. pneumophila-specific lymphoproliferation, indicating a deficiency in cell-mediated immunity. Normal mouse splenocytes treated in vitro with THC and pokeweed mitogen showed suppressed production of gamma interferon, a cytokine associated with Th1 cells, but increased production of interleukin 4, a cytokine produced by Th2 cells. Splenocytes from THC-treated mice, stimulated in vitro with either pokeweed mitogen or anti-CD3 antibodies, also produced less gamma interferon, indicating less Th1 activity in these mice. These results suggest that THC decreases the development of anti-L. pneumophila immunity by causing a change in the balance of Th1 and Th2 activities.

Animals↗
PubMed1994

A rapid colorimetric assay for evaluating Legionella pneumophila growth in macrophages in vitro.

A rapid colorimetric technique for in vitro quantitation of Legionella pneumophila intracellular proliferation in macrophages is described. The assay is based on the electron transport activity of metabolically active L. pneumophila. The yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is cleaved by the mitochondrial activity of viable L. pneumophila, forming a dark formazan derivative with an absorption spectrum different from that of the native compound. The MTT method for measuring intracellular growth of L. pneumophila closely correlated with the CFU assay. The ability of macrophages from the A/J mouse strain to support intracellular growth of L. pneumophila and the ability of desferrioxamine to restrict L. pneumophila intracellular proliferation were confirmed by both methods. The MTT assay offers the advantages of rapidity, simplicity, and cost efficiency over the CFU assay, since it can be performed in the same flat-bottom microtiter plate with measurement in an enzyme-linked immunosorbent assay reader, allowing efficient processing of large numbers of samples.

Animals↗