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T W Klein

Publications and source records attributed to T W Klein.

At least 37 records · Page 2Linked to original sources
PubMed1998

Marijuana, immunity and infection.

The influence of marijuana cannabinoids on immune function has been examined extensively over the last 25 yr. Various experimental models have been used employing drug-abusing human subjects, experimental animals exposed to marijuana smoke or injected with cannabinoids, and in vitro models employing immune cell cultures treated with various cannabinoids. For the most part, these studies suggest that cannabinoids modulate the function of T and B lymphocytes as well as NK cells and macrophages. In addition to studies examining cannabinoid effects on immune cell function, other reports have documented that these substances modulate host resistance to various infectious agents. Viruses such as herpes simplex virus and murine retrovirus have been studied as well as bacterial agents such as members of the genera Staphylococcus, Listeria, Treponema, and Legionella. These studies suggest that cannabinoids modulate host resistance, especially the secondary immune response. Finally, a third major area of host immunity and cannabinoids is that involving drug effects on the cytokine network. Employing in vivo and in vitro models, it has been determined that cannabinoids modulate the production and function of acute phase and immune cytokines as well as modulate the activity of network cells such as macrophages and T helper cells, Th1 and Th2. These results are intriguing and demonstrate that under certain conditions, cannabinoids can be immunomodulatory and enhance the disease process. However, more studies are needed to determine both the health risk of marijuana abuse and the role of the cannabinoid receptor/ligand system in immune regulation and homeostasis.

Animals↗
PubMed1998

Cannabinoid receptors and the cytokine network.

Splenocyte cultures from BALB/c mice were treated with THC and mitogen and shown to produce less Th1 cytokine, IFN gamma, and more Th2 cytokines, IL-4 and IL-10. This suggested that drug treatment caused a shift in the development of Th1 and Th2 cells. In studies designed to look at molecular mechanisms, the CBI antagonist, SR141716A, did not attenuate the THC enhancement of IL-4 production, but pertussis toxin attenuated the drug effect and the CB2 agonist, JWH-051, increased IL-4 production similar to THC. These results suggest that cannabinoids may increase Th2 development and IL-4 production in cultured immune cells through the activity of the CB2 receptor subtype. Studies are currently in progress to further define the molecular and cellular mechanisms involved.

Adjuvants, Immunologic↗
PubMed1998

Cannabinoid receptor agonists enhance syncytia formation in MT-2 cells infected with cell free HIV-1MN.

Marijuana and other drugs have been suggested to act as cofactors for HIV infection. Interestingly, delta 9-THC has been shown to upregulate NF kappa B, a transcription factor utilized by HIV. Therefore, it was of interest to investigate whether cannabinoids can modulate HIV infection and replication. Initially, we tested for evidence of receptor expression by examining for receptor mRNA in various cell lines used to study HIV infection and replication. Cellular RNA was isolated from SupT, and H9, H9MN, and MT-2 cells and RT-PCR was performed. Results showed that, although all of the cell lines tested were positive for CB2 mRNA, only the MT-2 cells also expressed CBI mRNA. Since the MT-2 cells expressed both CBI and CB2 receptor mRNA, we next wanted to determine whether different cannabinoid receptor agonists such as CP-55,940, delta 9-THC, WIN-55,212-2, and WIN-55,212-3 influenced infection of these cells by cell free HIV-1MN. Infectivity assays were performed where MT-2 cells were incubated with drug and cell free virus for 90 min, the free virus washed off, and the cells incubated further, and checked for virus growth by syncytia formation. It was found that the drugs significantly increased syncytia formation when MT-2 cells were cultured in the presence of both drug and cell free HIV-1MN. In conclusion, of the cell lines tested, only the MT-2 cells were positive for both CB1 and CB2 mRNA. In addition, since syncytia formation is an indication of virus infection and cytopathicity it was concluded that cannabimimetic drugs may enhance HIV-1 infection of susceptible cells.

Cell Line↗
PubMed1998

Delta9-tetrahydrocannabinol induces apoptosis in macrophages and lymphocytes: involvement of Bcl-2 and caspase-1.

Apoptosis is programed cell death characterized by certain cellular changes and regulated by various gene products including Bcl-2 and caspase-1. The marijuana cannabinoid, Delta9tetrahydrocannabinol (THC), has been reported to suppress in culture the proliferation of splenocytes and increase the release of IL-1 from macrophages; however, the mechanisms of these effects remain unclear. Because cannabinoids have also been reported to induce apoptosis and because the release of IL-1 and suppression of lymphoproliferation are related to apoptosis, we tested for the induction of apoptosis by THC in murine immune cell cultures. Splenocytes cultured with Con A for up to 24 hr showed evidence of DNA fragmentation determined by gel electrophoresis, terminal deoxynucleotide transferase-mediated dUTP-fluorescein nick end labeling and 3H-thymidine labeling and THC (15-30 microM) treatment increased fragmentation under these conditions. Resident peritoneal macrophages cultured with lipopolysaccharides showed no obvious fragmentation unless they were also treated with THC. Time course studies examining DNA fragmentation and cell membrane integrity (assessed by dye exclusion) showed that fragmentation preceded membrane damage indicating that THC induced apoptosis rather than cell necrosis. In addition, THC treatment of splenocytes resulted in a decrease of Bcl-2 mRNA and protein as measured by Northern and Western blotting, respectively, and the drug induced apoptosis was blocked by the caspase inhibitor, Ac-Tyr-Val-Ala-L-aspartic acid aldehyde. These data suggest that THC treatment of cultured immune cells induces apoptosis through the regulation of Bcl-2 and caspase activity.

Animals↗
PubMed1997

Differential effects of granulocyte/macrophage colony-stimulating factor (GM-CSF) in enhancing macrophage resistance to Legionella pneumophila vs Candida albicans.

It has been reported that granulocyte/macrophage colony-stimulating factor (GM-CSF), one of the hemopoietic growth factors which regulates the function of phagocytic cells, is a potent activator of cultured macrophages and induces antimicrobial activities as well as differentiation of precursor cells. In this study, we examined the ability of recombinant murine GM-CSF to activate mouse peritoneal macrophages to restrict the growth of two different microorganisms, Candida albicans and Legionella pneumophila, both of which are important opportunistic pathogens in an immunocompromised host. Treatment of thioglycollate-elicited BDF1 mouse macrophages with GM-CSF for 24 hr enhanced the anti-C. albicans activity of the macrophages in terms of inhibiting growth of the fungi. Reactive oxygen (H2O2) and IL-1 production by the macrophages were also enhanced by treatment with GM-CSF. However, no enhancement of anti-L. pneumophila activity of macrophages obtained from either susceptible A/J or resistant BDF1 mice to L. pneumophila infection after treatment with up to 1000 units/ml GM-CSF was observed under the same conditions. When the treatment time was extended to 72 hr. GM-CSF was still unable to induce anti-L. pneumophila activity. As a control study, treatment with recombinant IFN-gamma enhanced both anti-Candida and anti-Legionella activity in cultured macrophages under the same conditions used in the GM-CSF study. Measurement of cellular iron content revealed the low iron content in IFN-gamma-treated macrophages, but no decrease of iron in GM-CSF-treated macrophages compared with the control group, indicating a possible involvement of iron as a key factor in anti-L. pneumophila activity. Thus, the results of the study show that GM-CSF activation of elicited peritoneal macrophages is selective with regard to the type of antimicrobial activity induced.

Animals↗
PubMed1997

Induction of interleukin-2 receptor alpha gene by delta9-tetrahydrocannabinol is mediated by nuclear factor kappaB and CB1 cannabinoid receptor.

Previously, we reported that the cannabinoid delta9-tetrahydrocannabinol (THC) increased the expression of interleukin-2 (IL-2) receptor (R) alpha and beta proteins and mRNAs in NKB61A2 cells, but decreased the level of the gamma-chain message. The drug increased beta-chain message stability rather than increased transcription. In the present study, we examined the mechanism responsible for the drug-induced increase in alpha-chain message in NKB61A2 cells. Nuclear run-on and mRNA stability studies showed THC increased the level of alpha gene transcription but had no effect on mRNA stability. Because expression of this gene is regulated by nuclear factor (NF)-kappaB, we next tested the drug effect on the nuclear level of this protein using the electromobility shift assay. These studies showed a drug-induced increase in NF-kappaB activity. To link the increased nuclear factor activity with the THC-induced increase in IL-2R alpha message, antisense oligodeoxynucleotides were used to inhibit expression of the RelA component of NF-kappaB. These results showed anti-RelA antisense eliminated the cannabinoid-induced upregulation of both alpha mRNA and RelA protein. Furthermore, inhibition of the cannabinoid receptor type 1 with antisense oligomers also eliminated the drug effect on the alpha message. These results suggest that THC treatment of NKB61A2 cells increases IL-2R alpha gene transcription by increasing the nuclear level of NF-kappaB through a mechanism involving cannabinoid receptor type 1 expression.

Animals↗
PubMed1997

Involvement of mannose receptor in cytokine interleukin-1beta (IL-1beta), IL-6, and granulocyte-macrophage colony-stimulating factor responses, but not in chemokine macrophage inflammatory protein 1beta (MIP-1beta), MIP-2, and KC responses, caused by attachment of Candida albicans to macrophages.

The production of chemotactic cytokines (chemokines) and other cytokines by macrophages in response to fungal infection is thought to be critical during the course of candidiasis. However, the mechanism of cytokine synthesis by macrophages in response to fungi is not well understood. Therefore, the response of macrophages to Candida albicans was examined in terms of receptor-mediated chemokine and other cytokine mRNA induction. Attachment of C. albicans to murine thioglycollate-elicited peritoneal macrophages induced increased mRNA levels of the cytokines interleukin-1beta (IL-1beta), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and the chemokines macrophage inflammatory protein 1beta (MIP-1beta), MIP-2, and KC (a member of the platelet factor 4 neutrophil chemoattractant family), as determined by quantitative reverse transcription-PCR. However, treatment of macrophages with alpha-methyl-D-mannoside significantly reduced the cytokine GM-CSF response to C. albicans but did not affect the chemokine MIP-2 response. Antisense oligodeoxynucleotide (ODN) to mannose receptor (MR) mRNA inhibited the expression and function of MR in macrophages as determined by Western blot analysis and 125I-labeled mannose-bovine serum albumin (BSA) binding, and also inhibited the elevation of cytokine IL-1beta, IL-6, and GM-CSF mRNA levels induced by C. albicans attachment. Elevation of chemokine MIP-1beta, MIP-2, and KC mRNA levels induced by C. albicans was not affected in macrophages whose MR expression was suppressed by antisense ODN treatment. Furthermore, IL-4 treatment of macrophages, which up-regulated MR expression as determined by Western blot analysis and fluorescein isothiocyanate-labeled mannose-BSA uptake, enhanced the level of cytokine GM-CSF mRNA induced by C. albicans but not the level of the chemokine MIP-2 mRNA. These results indicate that selected cytokine responses of macrophages to C. albicans are mediated by MR, while some chemokine responses may be mediated by other receptors.

Animals↗
PubMed1996

Immunoregulatory role of nitric oxide in Legionella pneumophila-infected macrophages.

Nitric oxide (NO) is an intercellular messenger molecule produced by a variety of cells, including macrophages. However, the role of NO in infection, especially its immunological role, is poorly understood. In the present study, the role of NO in Legionella pneumophila-infected macrophages was examined. Whereas infection of mouse macrophages in vitro with L. pneumophila did not induce detectable NO, when the macrophages were primed with interferon-gamma (IFN-gamma), the treated macrophages markedly inhibited bacterial replication and produced a large amount of NO. Treatment with NO inhibitors, such as NG-monomethyl-L-arginine (L-MMA) or aminoguanidine, as well as culture in arginine-free medium, significantly inhibited NO production; however, the anti-L. pneumophila activity induced by IFN-gamma was not diminished. Examination of cytokine levels in L. pneumophila-infected macrophages primed with IFN-gamma revealed a moderate increase of interleukin-6 (IL-6) production; however, inhibition of NO by L-MMA markedly increased IL-6 production. Reconstitution of NO in the L. pneumophila-infected macrophages primed with IFN-gamma and treated with L-MMA to inhibit endogenous NO production following addition of sodium nitroprusside reduced IL-6 production to normal levels. The levels of IL-6 mRNA in L-MMA-treated macrophages were the same as in nontreated macrophages, as demonstrated by quantitative RT-PCR. Thus, these results indicate that NO may regulate IL-6 production independently of its role in antimicrobial function in L. pneumophila-infected macrophages and their immunoregulation on IL-6 production may be due to a post-transcriptional mechanism.

Animals↗
PubMed1996

Injection of complementary DNA encoding interleukin-12 inhibits tumor establishment at a distant site in a murine renal carcinoma model.

Interleukin (IL-12) protein has been shown to elicit diverse immunological responses and potent antitumor activity. We demonstrate here that intradermal injection of IL-12 cDNA induces systemic biological effects characteristic of the cytokine in vivo. Intradermal injection of IL-12 cDNA resulted in local expression of IL-12 mRNA, which correlated with a 10-fold increase in natural killer activity and a 3-4-fold increase in anti-CD3-induced IFN-gamma production in cultured splenocytes. Furthermore, when challenged with Renca tumor cells at a distant site, the day of tumor emergence was significantly delayed, and tumor growth was reduced in mice that received IL-12 cDNA, compared to mice given injections of plasmid vector alone. A number of the mice receiving IL-12 cDNA injections remained tumor free months after tumor challenge. In contrast to mice receiving recombinant IL-12 protein, no splenomegaly was detected when natural killer activity was significantly induced in mice receiving injections of IL-12 cDNA. Because purified plasmid DNA is more economical to prepare and has a longer shelf-life than recombinant proteins, and intradermal administration of cDNA encoding IL-12 did not cause splenomegaly, our findings suggest that the in vivo injection of cDNA encoding IL-12 may be a less toxic and more cost-effective alternative to IL-12 protein therapy in some clinical or experimental therapeutic applications.

Animals↗
PubMed1996

Legionella pneumophila heat-shock protein-induced increase of interleukin-1 beta mRNA involves protein kinase C signalling in macrophages.

Heat-shock proteins (hsp) are chaperon molecules important in protein folding and assembly. Furthermore, they may have functions in immunoregulatory processes, like T-cell stimulation and antigen presentation, which are not yet fully understood. It has been shown that several hsp of various species and family derivations modulate functions in macrophage immunity by directly increasing cytokine production. In the present study we showed that the 60,000 MW hsp of Legionella pneumophila (Lp-hsp 60) increased cellular steady-state levels of interleukin-1 beta (IL-1 beta) mRNA measured by quantitative reverse transcription-polymerase chain reaction and Northern blotting as well as IL-1 secretion, when added to cultures of thioglycollate-elicited mouse peritoneal macrophages in vitro. The level of mRNA increased in a dose-dependent manner with a minimum effective concentration of 0.5 microgram/ml and peaked 3 hr after stimulation. Lp-hsp 60-coated latex beads also increased IL-1 beta mRNA levels in the presence of cytochalasin D, which inhibits bead uptake but permits binding, indicating that binding to the macrophage surface was sufficient for induction. Accumulation of IL-1 beta mRNA was completely blocked by pretreatment with the protein kinase C (PKC) inhibitor, H7, but not decreased by prior treatment with cycloheximide. The cell lysates of macrophages stimulated with Lp-hsp 60 showed an increased PKC activity measured by phosphorylation of PKC pseudosubstrate. The IL-1 bioactivity in culture supernatants after 24 hr of stimulation with Lp-hsp 60 was increased in a dose-dependent manner but at hsp concentrations in excess of those needed to increase mRNA. Thus, the present study demonstrates that Lp-hsp 60 rapidly increases the steady-state level of IL-1 beta mRNA, possibly through a cell surface receptor system involving a PKC-dependent signalling pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Induction of cytokine granulocyte-macrophage colony-stimulating factor and chemokine macrophage inflammatory protein 2 mRNAs in macrophages by Legionella pneumophila or Salmonella typhimurium attachment requires different ligand-receptor systems.

The attachment of bacteria to macrophages is mediated by different ligands and receptors and induces various intracellular molecular responses. In the present study, induction of cytokines and chemokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage inflammatory protein 2 (MIP-2), was examined, following bacterial attachment, with regard to the ligand-receptor systems involved. Attachment of Legionella pneumophila or Salmonella typhimurium to cultured mouse peritoneal macrophages increased the steady-state levels of cellular mRNAs for the cytokines interleukin 1beta (IL-1beta), IL-6, and GM-CSF as well as the chemokines MIP-1beta, MIP-2, and KC. However, when macrophages were treated with alpha-methyl-D-mannoside (alphaMM), a competitor of glycopeptide ligands, induction of cytokine mRNAs was inhibited, but the levels of chemokine mRNAs were not. Pretreatment of the bacteria with fresh mouse serum enhanced the level of GM-CSF mRNA but not the level of MIP-2 mRNA. In addition, serum treatment reduced the inhibitory effect of alphaMM on GM-CSF mRNA. These results indicate that bacterial attachment increases the steady-state levels of the cytokine and chemokine mRNAs tested by at least two distinct receptor-ligand systems, namely, one linked to cytokine induction and involving mannose or other sugar residues and the other linked to chemokine induction and relatively alphaMM insensitive. Furthermore, opsonization with serum engages other pathways in the cytokine response which are relatively independent of the alphaMM-sensitive system. Regarding bacterial surface ligands involved in cytokine mRNA induction, evidence is presented that the flagellum may be important in stimulating cytokine GM-CSF message but not chemokine MIP-2 message. Analysis of cytokine GM-CSF and chemokine MIP-2 signaling pathways with protein kinase inhibitors revealed the involvement of calmodulin and myosin light-chain kinase in GM-CSF but not MIP-2 mRNA induction, adding further evidence that several distinct receptor systems are engaged during the process of bacterial attachment and induction of cytokines and chemokines, such as GM-CSF and MIP-2, respectively.

Animals↗
PubMed1996

Antifungal antibiotic benanomicin A increases susceptibility of Candida albicans to phagocytosis by murine macrophages.

Benanomicin A is an antifungal antibiotic produced by Actinomadura spadix. In the present study, we investigated the effect of benanomicin A on the phagocytosis of Candida albicans by murine peritoneal macrophages and on the cell-surface hydrophobicity (CSH) of C. albicans. Although pretreatment of macrophages with benanomicin A had no effect on the phagocytosis, addition of benanomicin A to the culture of macrophages and Candida cells increased the susceptibility of Candida cells to the phagocytosis by the macrophages. Pretreatment of Candida cells with benanomicin A also increased the susceptibility of Candida cells to the phagocytosis. When Candida cells were mixed with benanomicin A, the antibiotic bound irreversibly to Candida cells. These data suggest the possibility that the increased susceptibility of Candida cells to the phagocytosis is mediated by the binding of benanomicin A to Candida cells. Examination of physicochemical property of Candida cell surface showed that the CSH of Candida cells significantly decreased by the treatment with benanomicin A. Thus, binding of benanomicin A to Candida cells may induce biochemical/physicochemical alternation of the surfaces, so that they become more susceptible to phagocytosis by murine macrophages. These properties of benanomicin A, along with its antifungal activity, seem to be beneficial in the treatment of fungal infections.

Animals↗
PubMed1996

Cannabinoid receptor proteins are increased in Jurkat, human T-cell line after mitogen activation.

Brain-type cannabinoid receptor (CB1) mRNA has been demonstrated in several peripheral tissues; however, the function of this message is not clear. In the present study, we examined the levels of CB1 mRNA and receptor protein in stimulated immune cells to link message and protein expression with cell activation. Consistent with previous reports, immune cell lines from human and mouse were positive by reverse transcription-polymerase chain reaction for CB1 mRNA; however, one cell line, Jurkat, was only weakly positive. Mitogen activation of Jurkat cells, however, increased CB1 mRNA within 2 hr after stimulation and equilibrium binding studies, using Jurkat membranes, showed [3H]CP55,940 specific binding was negative early after mitogen stimulation but positive at 40 hr poststimulation. To investigate whether this observed increase in CB1 mRNA and specific binding activity was associated with expression of the CB1 protein, polyclonal antibodies were produced to a fusion protein consisting of glutathione S-transferase and a 342 amino acid portion (residues 33 through 374) of the CB1 protein. Western blotting analysis showed expression of several immunoreactive proteins on membranes from mitogen-activated Jurkat cells, but not on membranes from unstimulated cells. These results demonstrate a link between the level of CB1 mRNA and surface protein in activated immune cells, suggesting the possibility of a functional role of CB1 in immune cell activation.

Animals↗
PubMed1995

LPS inhibits the intracellular growth of Legionella pneumophila in thioglycolate elicited murine peritoneal macrophages by iron-dependent, tryptophan-independent, oxygen-independent, and arginine-independent mechanisms.

Thioglycolate-elicited murine macrophages from genetically susceptible A/J mice activated with lipopolysaccharide (LPS) and infected with Legionella pneumophila in vitro evince marked inhibition of intracellular growth of this bacterium. The mechanism of inhibition by LPS-activated macrophages in terms of replication of this intracellular pathogen is unclear. LPS activation of murine macrophages induced a downshift in transferrin receptor (TfR) expression and reduction in cellular iron content, and this was correlated with augmented intracellular growth of Legionella in the cells. When LPS-stimulated macrophages were first saturated with iron, partial reversion of L. pneumophila growth restriction was observed. However, an excess of exogenous L-tryptophan (Trp) did not reverse this growth inhibition, nor did supplementation of the macrophage culture medium with both iron and Trp. The antilegionella activity of the macrophages induced by LPS activation was independent of reactive oxygen intermediates (ROI), since the scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on growth restriction. Likewise, notwithstanding the ability of LPS-activated macrophages to synthesize reactive nitrogen intermediates (RNI), which was inhibited by L-arginine analogs NG-monomethyl-L-arginine and L-aminoguanidine), as well as by incubation in arginine-free medium, their ability to inhibit the intracellular replication of L. pneumophila was not affected. Thus, we conclude that LPS-activated macrophages inhibit the intracellular growth of L. pneumophila partially by iron-dependent, Trp-independent, and ROI- and RNI-independent mechanisms. We also suggest that additional unknown mechanisms are involved, since complete reversion was not obtained.

Animals↗
PubMed1995

Spatial learning impairment in mice infected with Legionella pneumophila or administered exogenous interleukin-1-beta.

The effect of interleukin-1 beta (IL 1 beta) on spatial learning was examined. In one experiment, C57BL/6 mice were given daily injections (100 ng/mouse) of recombinant murine IL1 beta prior to training on the Morris water maze. In another experiment, mice were infected with a sublethal dose of a gram-negative bacterium (Legionella pneumophila; Lp). Mice rendered ill by the infection were given either anti-IL1 beta antibodies (100 micrograms/mouse) or saline and then trained on the water maze. Results indicated that (1) exogenous IL1 beta blocked acquisition of spatial learning, (2) Lp infection attenuated learning on this task, and (3) neutralizing circulating IL1 beta in Lp-infected mice normalized learning despite the continuation of the illness. The data indicate that cognitive impairment may be a component of cytokine-mediated sickness behavior.

Animals↗