The peptidyl-prolyl isomerase Pin1 regulates granulocyte-macrophage colony-stimulating factor mRNA stability in T lymphocytes

Cytokine production is associated with both the normal and pathologic inflammatory response to injury. Previous studies have shown that the immunosuppressants cyclosporin A or FK506, which interact with the peptidyl-propyl isomerases cyclophilin A and FK506-binding protein (FKBP12), respectively, block cytokine expression. A third member of the peptidyl-propyl isomerase family, Pin1 is expressed by immune and other cells. Pin1 has been implicated in cell cycle progression, is overexpressed in human tumors, and may rescue neurons from tau-associated degeneration. However, the role of Pin1 in the immune system remains largely unknown. In this study, we analyze the role of Pin1 in GM-CSF expression by human PBMC and CD4+ lymphocytes. We show that Pin1 isomerase activity is necessary for activation-dependent, GM-CSF mRNA stabilization, accumulation, and protein secretion, but not non-AU-rich elements containing cytokine mRNAs, including TGF-beta and IL-4. Mechanistically, Pin1 mediated the association of the AU-rich element-binding protein, AUF1, with GM-CSF mRNA, which determined the rate of decay by the exosome.     

Human Immunodeficiency Virus Neurotropism: an Analysis of Viral Replication and Cytopathicity for Divergent Strains in Monocytes and Microglia

Productive replication of human immunodeficiency virus type 1 (HIV-1) in brain macrophages and microglia is a critical component of viral neuropathogenesis. However, how virus-macrophage interactions lead to neurological disease remains incompletely understood. Possibly, a differential ability of virus to replicate in brain tissue macrophages versus macrophages in other tissues underlies HIV-1 neurovirulence. To these ends, we established systems for the isolation and propagation of pure populations of human microglia and then analyzed the viral life cycles of divergent HIV-1 strains in these cells and in cultured monocytes by using identical viral inocula and indicator systems. The HIV-1 isolates included those isolated from blood, lung tissue, cerebrospinal fluids (CSF), and brain tissues of infected subjects: HIV-1_ADA and HIV-1_89.6 (from peripheral blood mononuclear cells), HIV-1_DJV and HIV-1_JR-FL (from brain tissue), HIV-1_SF162 (from CSF), and HIV-1_BAL (from lung tissue). The synthesis of viral nucleic acids and viral mRNA, cytopathicity, and release of progeny virions were assessed. A significant heterogeneity among macrophage-tropic isolates for infection of monocytes and microglia was demonstrated. Importantly, a complete analysis of the viral life cycle revealed no preferential differences in the abilities of the HIV-1 strains tested to replicate in microglia and/or monocytes. Macrophage tropism likely dictates the abilities of HIV-1 to invade, replicate, and incite disease within its microglial target cells.     

Role of the β-Chemokine Receptors CCR3 and CCR5 in Human Immunodeficiency Virus Type 1 Infection of Monocytes and Microglia

Human immunodeficiency virus type 1 (HIV-1) infection in mononuclear phagocyte lineage cells (monocytes, macrophages, and microglia) is a critical component in the pathogenesis of viral infection. Viral replication in macrophages serves as a reservoir, a site of dissemination, and an instigator for neurological sequelae during HIV-1 disease. Recent studies demonstrated that chemokine receptors are necessary coreceptors for HIV-1 entry which determine viral tropism for different cell types. To investigate the relative contribution of the β-chemokine receptors CCR3 and CCR5 to viral infection of mononuclear phagocytes we utilized a panel of macrophage-tropic HIV-1 strains (from blood and brain tissue) to infect highly purified populations of monocytes and microglia. Antibodies to CD4 (OKT4A) abrogated HIV-1 infection. The β chemokines and antibodies to CCR3 failed to affect viral infection of both macrophage cell types. Antibodies to CCR5 (3A9) prevented monocyte infection but only slowed HIV replication in microglia. Thus, CCR5, not CCR3, is an essential receptor for HIV-1 infection of monocytes. Microglia express both CCR5 and CCR3, but antibodies to them fail to inhibit viral entry, suggesting the presence of other chemokine receptors for infection of these cells. These studies demonstrate the importance of mononuclear phagocyte heterogeneity in establishing HIV-1 infection and persistence.     

CD4-independent infection of astrocytes by human immunodeficiency virus type 1: requirement for the human mannose receptor

Human immunodeficiency virus type 1 (HIV-1) infection occurs in the central nervous system and causes a variety of neurobehavioral and neuropathological disorders. Both microglia, the residential macrophages in the brain, and astrocytes are susceptible to HIV-1 infection. Unlike microglia that express and utilize CD4 and chemokine coreceptors CCR5 and CCR3 for HIV-1 infection, astrocytes fail to express CD4. Astrocytes express several chemokine coreceptors; however, the involvement of these receptors in astrocyte HIV-1 infection appears to be insignificant. In the present study using an expression cloning strategy, the cDNA for the human mannose receptor (hMR) was found to be essential for CD4-independent HIV-1 infectivity. Ectopic expression of functional hMR rendered U87.MG astrocytic cells susceptible to HIV-1 infection, whereas anti-hMR serum and hMR-specific siRNA blocked HIV-1 infection in human primary astrocytes. In agreement with these findings, hMR bound to HIV-1 virions via the abundant and highly mannosylated sugar moieties of HIV-1 envelope glycoprotein gp120 in a Ca(2+)-dependent fashion. Moreover, hMR-mediated HIV-1 infection was dependent upon endocytic trafficking as assessed by transmission electron microscopy, as well as inhibition of viral entry by endosomo- and lysosomotropic drugs. Taken together, these results demonstrate the direct involvement of hMR in HIV-1 infection of astrocytes and suggest that HIV-1 interaction with hMR plays an important role in HIV-1 neuropathogenesis.     

Cyclosporin A and FK506 mediate differential effects on T cell activation in vivo.

Modified limiting dilution analysis techniques were used to evaluate the effects of the immunosuppressants cyclosporin A (CsA) and FK506 on alloantigen-induced T cell activation in vivo. Treatment of sponge matrix allograft recipients with either CsA or FK506 inhibited lymphocytic infiltration of the allograft, a process thought to be dependent on local lymphokine production. In addition, both immunosuppressants markedly reduced the absolute number of lymphocytes recovered from the draining lymph nodes (LN) and prevented CTL activation in the LN. However, Ag-primed helper T lymphocytes (HTL) were present in the draining LN of sponge allograft recipients treated with CsA, but not in recipients treated with FK506. T cell depletion experiments were performed to determine the phenotype of primed HTL in the LN of untreated and CsA-treated sponge allograft recipients. In untreated sponge allograft recipients, CD4+ and CD8+ Ag-primed HTL were present in the draining LN in equivalent numbers. In contrast, the majority of primed HTL in the LN of CsA-treated sponge allograft recipients were CD8+, rather than CD4+ T cells. These observations indicate that CsA and FK506 exert distinct in vivo effects at the level of HTL priming, and CD4+ and CD8+ HTL exhibit differential sensitivity to CsA in vivo.     

Effects of cyclosporin A, FK506 and rapamycin on calcineurin phosphatase activity in mouse brain

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase expressed at high levels in brain. The immunosuppressive drugs cyclosporin A and FK506, but not rapamycin are specific inhibitors of calcineurin, the inhibitory effects of which have been elucidated in the immune system. Here by using these compounds as inhibitors, we assayed the enzyme in mouse brain after injection of 12.5 nmol cyclosporin A, FK506, or rapamycin into the left lateral ventricle of mouse brain. Data from calcineurin activity assay suggest that infusion of cyclosporin A or FK506, rather than rapamycin inhibited calcineurin activity in brain and in a substrate noncompetitive manner, which is revealed by the in vitro enzyme kinetic analysis. Cyclosporin A or FK506 injected into brains also affected the inhibitory effects of cyclosporin A or FK506 added to brain extracts on calcineurin activity. The results may be ascribed to the decreased free immunophilin in brain after infusion of corresponding immunosuppressant, or the fact that two immunophilin-immunosuppressant complexes have not completely identical interaction sites on calcineurin.     

Inhibitory effect of FK 506 and cyclosporin A on nitric oxide production by LPS-treated cultured rat macrophages

We analyzed the effect of FK 506 on the production of nitric oxide by macrophages. Isolated rat peritoneal macrophages were cultured for 24 h with or without lipopolysaccharide (LPS) (5 microg/ml) and in the absence or presence of FK 506 (0.1 and 1 microg/ml). The concentration of NO2- in culture supernatants was taken as a measure of nitric oxide production. FK 506 (0.1 and 1 microg/ml) reduced the LPS-induced increase of NO2- levels by 68% and 81%, respectively. The impact of cyclosporin A (CsA) was studied in order to compare their effects. CsA (0.1 and 1 microg/ml) decreased the levels of nitrites by 39% and 69%, respectively. The results obtained suggest that both immunosuppressive drugs exhibit a dose-dependent inhibitory effect on nitric oxide production and that FK 506 is a more potent agent than CsA in this respect.     

Nuclear factor of activated T cells is a driving force for preferential productive HIV-1 infection of CD45RO-expressing CD4+ T cells

Human immunodeficiency virus type-1 (HIV-1) preferentially replicates in CD4-expressing T cells bearing a "memory" (CD45RO+) rather than a "naive" (CD45RA+/CD62L+) phenotype. Yet the basis for the higher susceptibility of these cells to HIV-1 infection remains unclear. Because the nature of the CD45 isoform itself can affect biochemical events in T cells, we set out to determine whether these isoforms could differently modulate HIV-1 long terminal repeat (LTR) activity and thereby replication. Through the use of CD4+ Jurkat T cells specifically expressing distinct CD45 isoforms (i.e. CD45RABC or CD45RO), we demonstrated that a difference in CD45 isoform expression conferred preferential replication of HIV-1 to CD45RO-expressing T cell clones following a physiological CD3/CD28 stimulation. Closer analysis indicated that higher HIV-1 LTR activation levels were consistently observed in CD45RO-positive cells, which was paralleled by more pronounced nuclear factor of activated T cells (NFAT) activation in these same cells. Specific involvement of NFAT1 was revealed in studied Jurkat clones by mobility shift analyses. In addition, preferential activation of the LTR and viral replication in CD45RO T cells was FK506- and cyclosporin A-sensitive. These results underscore the importance of NFAT in HIV-1 regulation and for the first time identify the role of the CD45 isoform in limiting productive HIV-1 replication to the human CD4 memory T cell subset.     

Enigma of HIV-1 latent infection in astrocytes: an in-vitro study using protein kinase C agonist as a latency reversing agent

Purging HIV-1 to cure the infection in patients undergoing suppressive antiretroviral therapy requires targeting all possible viral reservoirs. Other than the memory CD4⁺ T cells, several other HIV-1 reservoirs have been identified. HIV-1 infection in the brain as a reservoir is well documented, but not fully characterized. There, microglia, perivascular macrophages, and astrocytes can be infected by HIV-1. HIV-1 infection in astrocytes has been described as a nonproductive and primarily a latent infection. Using primary human astrocytes, we investigated latent HIV-1 infection and tested phorbol 12-myristate 13-acetate (PMA), a protein kinase C agonist, as an HIV-1-latency- reversing agent in infected astrocytes. Chloroquine (CQ) was used to facilitate initial HIV-1 escape from endosomes in astrocytes. CQ significantly increased HIV-1 infection. But treatment with PMA or viral Tat protein was similar to untreated HIV-1-infected astrocytes. Long-term follow-up of VSV-envelope-pseudotyped HIV-1 infected astrocytes showed persistent infection for 110 days, indicating the active state of the virus.     

Macrophage tropism of human immunodeficiency virus type 1 isolates from brain and lymphoid tissues predicts neurotropism independent of coreceptor specificity

The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.     

Effect of FK506 and cyclosporine A on the expression of BDNF, tyrosine kinase B and p75 neurotrophin receptors in astrocytes exposed to simulated ischemia in vitro

We investigated whether the immunosuppressive drugs, FK506 and cyclosporine A, increase BDNF protein and/or mRNA expression in ischemic astrocytes and if an increase could be related to changes in the nuclear expression of p-CREB, p-Erk1/2 and p-Akt. The influence of these immunosuppressants on protein and mRNA levels of TrkB and p75^NTR receptors was also examined. On day 21, cultures of rat astrocytes were subjected to ischemic conditions simulated in vitro (combined oxygen glucose deprivation, OGD) for 8 h and exposed to FK506 (10-1000 nM) and cyclosporine A (0.25-10 μM). FK506 and cyclosporine A (at 1000 nM and 0.25 μM, respectively) stimulated the expression and release of BDNF in cultured rat cerebral cortical astrocytes exposed to OGD. The immunosuppressants at these doses simultaneously increased p-CREB and p-Erk1/2 expression in the nuclear fraction of astrocytes. The results RT-PCR and Western blot analysis provided further evidence of a modulating influence of the drugs on the expression of trkB and p75^NTR genes and their protein products in ischemic astrocytes.     

Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands.

The peptidyl-prolyl isomerases FKBP12 and cyclophilin A (immunophilins) form complexes with the immunosuppressants FK506 and cyclosporin A that inhibit the phosphatase calcineurin. With the yeast two hybrid system, we detect complexes between FKBP12 and the calcineurin A catalytic subunit in both the presence and absence of FK506. Mutations in FKBP12 surface residues or the absence of the calcineurin B regulatory subunit perturb the FK506-dependent, but not the ligand-independent, FKBP12-calcineurin complex. By affinity chromatography, both FKBP12 and cyclophilin A bind calcineurin A in the absence of ligand, and FK506 and cyclosporin A respectively potentiate these interactions. Both in vivo and in vitro, the peptidyl-prolyl isomerase active sites are dispensable for ligand-independent immunophilin-calcineurin complexes. Lastly, by genetic analyses we demonstrate that FKBP12 modulates calcineurin functions in vivo. These findings reveal that immunophilins interact with calcineurin in the absence of exogenous ligands and suggest that immunosuppressants may take advantage of the inherent ability of immunophilins to interact with calcineurin.     

Calcineurin enhances MEF2 DNA binding activity in calcium-dependent survival of cerebellar granule neurons.

Myocyte enhancer factor 2 (MEF2) has been shown recently to be necessary for mediating activity-dependent neuronal survival. In this study, we show that calcium signals regulate MEF2 activity through a serine/threonine phosphatase calcineurin. In cultured primary cerebellar granule neurons, the electrophoretic mobility of MEF2A protein was sensitive to the level of extracellular potassium chloride (KCl) and depolarizing concentrations of KCl led to hypophosphorylation of the protein. The specific inhibitors of calcineurin cyclosporin A (CsA) and FK506 could overcome KCl-dependent MEF2A hypophosphorylation. The effects of CsA and FK506 were KCl specific as they had little effect on MEF2A phosphorylation when granule neurons were cultured in the presence of full media. Hyperphosphorylation of MEF2A led to the loss of its DNA binding activity as determined by DNA mobility shift assay. Consistent with this, CsA/FK506 also inhibited MEF2-dependent reporter gene expression. These findings demonstrate that regulation of MEF2A by calcium signals requires the action of protein phosphatase calcineurin. By maintaining MEF2A in a hypophosphorylated state, calcineurin enhances the DNA binding activity of MEF2A and therefore maximizes its transactivation capability. The identification of MEF2 as a novel target of calcineurin may provide in part a biochemical explanation for the therapeutic and toxic effects of immunosuppressants CsA and FK506.     

Molecular mechanisms of neuroprotective action of immunosuppressants--facts and hypotheses

Cyclosporin A (CsA) and FK506 (Tacrolimus) are short polypeptides which block the activation of lymphocytes and other immune system cells. Immunosuppressants exert neuroprotective and neurotrophic action in traumatic brain injury, sciatic nerve injury, focal and global ischemia in animals. Their neuroprotective actions are not understood and many hypotheses have been formed to explain such effects. We discuss a role of drug target - calcineurin in neuroprotective action of immunosuppressants. Protein dephosphorylation by calcineurin plays an important role in neuronal signal transduction due to its ability to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In vitro FK506 protects cortex neurons from NMDA-induced death, augments NOS phosphorylation inhibiting its activity and NO synthesis. However, in vivo experiments demonstrated that FK506 in neuroprotective doses did not block excitotoxic cell death nor did it alter NO production during ischemia/reperfusion. Tissue damage in ischemia is the result of a complex pathophysiological cascade, which comprises a variety of distinct pathological events. Resident non-neuronal brain cells respond rapidly to neuronal cell death and may have both deleterious and useful role in neuronal damage. There is increasing evidence that reactive gliosis and post-ischemic inflammation involving microglia contribute to ischemic damage. We have demonstrated that FK506 modulates hypertrophic/proliferative responses and proinflammatory cytokine expression in astrocytes and microglia in vitro and in focal transient brain ischemia. Our findings suggest that astrocytes and microglia are direct targets of FK506 and modulation of glial response and inflammation is a possible mechanism of FK506-mediated neuroprotection in ischemia.     

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.     

Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.