Increasing evidence suggests that CD45, a transmembrane protein tyrosine phosphatase, is an important modulator of macrophage activation. Microglia, resident brain macrophages, express CD45 and proliferate under pathologic conditions. In this study, we examined the role of CD45 in modulating GM-CSF-induced proliferation and signal transduction in primary human microglial cultures. Soluble, but not immobilized anti-CD45RO induced tyrosine phosphatase activity and inhibited GM-CSF-induced microglial proliferation. Microglial proliferation was also inhibited by PP2 (Src inhibitor), LY294002 (PI3K inhibitor), and U0126 (MEK inhibitor). GM-CSF induced phosphorylation of Jak2, Stat5, Hck (the myeloid-restricted Src kinase), Akt, Stat3, and Erk MAPKs in microglia. Of these, anti-CD45RO inhibited phosphorylation of Hck and Akt, and PP2 inhibited phosphorylation of Hck and Akt. In a macrophage cell line stably overexpressing wild-type or kinase-inactive Hck, GM-CSF increased proliferation of the control (empty vector) and wild-type but not kinase-inactive cells, and this was inhibited by anti-CD45RO. Together, these results demonstrate that, in macrophages, Hck tyrosine kinase is activated by GM-CSF, and that Hck plays a pivotal role in cell proliferation and survival by activating the PI3K/Akt pathway. Ab-mediated activation of macrophage and microglial CD45 tyrosine phosphatase may have therapeutic implications for CNS inflammatory diseases.

Bone marrow-derived mononuclear cells (BMNCs) have been shown to effectively treat ischemic cardiovascular diseases. Because diabetic neuropathy (DN) is causally associated with impaired angiogenesis and deficiency of angiogenic and neurotrophic factors in the nerves, we investigated whether DN can be ameliorated by local injection of BMNCs. Severe peripheral neuropathy, characterized by a significant decrease in the motor and sensory nerve conduction velocities (NCVs), developed 12 weeks after the induction of diabetes with streptozotocin in rats. The injection of BMNCs restored motor and sensory NCVs to normal levels and significantly improved vascular density and blood flow in diabetic nerves over 4 weeks. Fluorescent microscopic observation revealed that DiI-labeled BMNCs preferentially engrafted in sciatic nerves. Whole-mount fluorescent imaging and confocal microscopic evaluation demonstrated that many of the BMNCs localized following the course of the vasa nervorum in close proximity to blood vessels without incorporation into vasa nervorum as endothelial cells at a detectable level. Real-time reverse transcription-polymerase chain reaction analysis showed that the levels of angiogenic and neurotrophic factors were significantly increased in the nerves by BMNC injection. Local transplantation of BMNCs improved experimental DN by augmenting angiogenesis and increasing angiogenic and neurotrophic factors in peripheral nerves. These findings suggest that BMNC transplantation may represent a novel therapeutic option for treating DN.

Microglia, the resident brain macrophages, are the principal cells involved in the regulation of inflammatory and antimicrobial responses in the CNS. Interferon-b (IFNb) is an antiviral cytokine induced by viral infection or following non-specific inflammatory challenges of the CNS. Because of the well-known anti-inflammatory properties of IFNb, it is also used to treat multiple sclerosis, an inflammatory CNS disease. Despite the importance of IFNb signaling in CNS cells, little has been studied, particularly in microglia. In this report, we investigated the molecular mechanisms underlying IFNbinduced b-chemokine expression in primary human fetal microglia. Multiple signaling cascades are activated in microglia by IFNb, including nuclear factor-jB (NF-jB), activator protein-1 (AP-1) and Jak/Stat. IFNb induced IjBa degradation and NF-jB (p65:p50) DNA binding. Inhibition of NF-jB by either adenoviral transduction of a super repressor IjBa, or an antioxidant inhibitor of NF-jB reduced expression of the b-chemokines, regulated upon activation, normal T-cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)-1b. IFNb also induced phosphorylation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase, and the MAP kinase kinase 1 (MEK1) inhibitor PD98059 dose-dependently inhibited b-chemokine mRNA and protein expression. PD98059 did not inhibit NF-jB binding, demonstrating that ERK was not responsible for NF-jB activation. Two downstream targets of ERK were identified in microglia: AP-1 and Stat1. IFNb induced AP-1 nuclear binding activity in microglia and this was suppressed by PD98059. Additionally, IFNb induced Stat1 phosphorylation at both tyrosine 701 (Y701) and serine 727 (S727) residues. S727 phosphorylation of Stat1, which is known to be required for maximal transcriptional activation, was inhibited by PD98059. Our results demonstrating multiple signaling cascades initiated by IFNb in primary human microglia are novel and have implications for inflammatory and infectious diseases of the CNS.

Macrophages and microglia are productively infected by HIV-1 and play a pivotal role in the pathogenesis of AIDS dementia. Although macrophages and microglia express CD45, a transmembrane protein tyrosine phosphatase, whether modulation of its activity affects human immunodeficiency virus type 1 (HIV-1) replication is unknown. Here, we report that of the five human CD45 isoforms, microglia express CD45RB and CD45RO (RB > RO) and treatment of microglia with a CD45 agonist antibody ␣CD45RO (

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CD45 is a membrane tyrosine phosphatase that modulates the development and function of hematopoietic cells including macrophages. Several isoforms of CD45 exist as a result of alternative splicing of the extracellular domain exons (A, B and C). Agonist antibodies to CD45 isoforms have been shown to suppress microglial activation, proliferation and HIV infection, suggesting its potential as a new therapeutic target. To determine the cell‐type and disease‐specific expression of CD45 isoforms in human CNS, sections of HIV‐1 encephalitis (HIVE) and controls (HIV‐ and HIV+) were studied by immunohistochemistry using CD45 exon‐specific (RA, RB, RC and RO) antibodies. The results showed that RA and RC expression was limited to rare lymphocytes, while RB expression was robust in microglia (both control and HIVE) and inflammatory cells (T cells and macrophages). RO was low in control microglia, but increased in HIVE. T cells and macrophages also expressed RO. Our study demonstrated that CD45 expression in the CNS is limited to two isoforms (RB and RO) and that RB is also expressed in resting ramified microglia. Targeting CD45RO with an antibody might be a therapeutic option for neuroinflammatory diseases.