FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W87068116> ?p ?o ?g. }

• W87068116 endingPage "1453" @default.
• W87068116 startingPage "1441" @default.
• W87068116 abstract "Human immunodeficiency virus (HIV) encephalitis is a prominent pathology seen in children infected with HIV. Immunohistochemical analyses of pediatric brain tissue showed distinct differences in expression of C-C chemokines and their receptors between children with HIV encephalitis and those with non-CNS-related pathologies. Evidence suggests that soluble factors such as HIV Tat released from HIV-infected cells may have pathogenic effects. Our results show Tat effects on chemokines and their receptors in microglia and astrocytes as well as chemokine autoregulation in these cells. These results provide evidence for the complex interplay of Tat, chemokines, and chemokine receptors in the inflammatory processes of HIV encephalitis and illustrate an important new role for chemokines as autocrine regulators. Human immunodeficiency virus (HIV) encephalitis is a prominent pathology seen in children infected with HIV. Immunohistochemical analyses of pediatric brain tissue showed distinct differences in expression of C-C chemokines and their receptors between children with HIV encephalitis and those with non-CNS-related pathologies. Evidence suggests that soluble factors such as HIV Tat released from HIV-infected cells may have pathogenic effects. Our results show Tat effects on chemokines and their receptors in microglia and astrocytes as well as chemokine autoregulation in these cells. These results provide evidence for the complex interplay of Tat, chemokines, and chemokine receptors in the inflammatory processes of HIV encephalitis and illustrate an important new role for chemokines as autocrine regulators. Chemokines are small molecular weight proteins involved in the processes of leukocyte recruitment and activation.1Luster A Chemotactic cytokines in inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3232) Google Scholar, 2Rollins B Chemokines.Blood. 1997; 90: 909-928Crossref PubMed Google Scholar There are several families of chemokines classified by the position of their N-terminal cysteines; C-X-C, C-C, C, and C-X3-C. The C-C chemokine family, including monocyte chemoattractant protein 1 (MCP-1) and the macrophage inflammatory proteins (MIP-1α and MIP-1β), serves primarily as chemoattractants for monocytes and T cells. These proteins function through binding of specific seven transmembrane domain spanning, G-protein-coupled receptors. These receptors bind chemokines within their family and the C-C chemokine receptor family is continually growing with approximately 10 receptors identified. Chemokine receptor binding within each family is somewhat promiscuous with MIP-1α binding CCR1 and CCR5, MIP-1β binding CCR5, and MCP-1 using the CCR2 receptor. Both chemokines and their receptors have been shown to play key roles in human immunodeficiency virus (HIV) infection and progression. Several chemokine receptors are co-factors with CD4 for the entry of HIV into host cells, the major receptors being CCR5 and CXCR4.3Feng Y Broder CC Kennedy PE Berger EA HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane domain, G-protein coupled receptor.Science. 1996; 272: 872-877Crossref PubMed Scopus (3621) Google Scholar, 4Alkhatib G Combadiere C Broder CC Feng Y Kennedy PE Murphy PM Berger EA CC CKR5: A RANTES, MIP-1α, MIP-1β receptor as a fusion cofactor for macrophage-tropic HIV-1.Science. 1996; 272: 1955-1958Crossref PubMed Scopus (2437) Google Scholar, 5Dragic T Litwin V Allaway GP Martin SR Huang Y Nagashima KA Cayanan C Maddon PJ Koup RA Moore JP Paxton WA HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.Nature. 1996; 381: 667-673Crossref PubMed Scopus (2811) Google Scholar, 6Choe H Farzan M Sun Y Sullivan N Rollins B Ponath PD Wu L Mackay CR LaRosa G Newman W Gerard N Gerard C Sodroski J The β-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.Cell. 1996; 85: 1135-1148Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar Chemokines have been shown to compete with HIV for binding of chemokine receptors and as such may play a role in controlling the spread of the virus within the host.7Cocchi F DeVico AL Garzino-Demo A Arya SK Gallo RC Lusso P Identification of RANTES, MIP-1α, and MIP-1β as the major HIV suppressive factors produced by CD8+ T cells.Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2620) Google ScholarA major complication of HIV infection, particularly in children, is encephalitis with approximately one-third of those infected with HIV developing HIV encephalitis and/or acquired immune deficiency syndrome dementia complex.8Kolson DL Lavi E Gonzalez-Scarano F The effects of HIV in the CNS.Adv Virus Res. 1998; 50: 1-47Crossref PubMed Scopus (123) Google Scholar Although much is known about the role of chemokines and their receptors in the pathogenesis of HIV infection, little is known of their role in HIV infection of the central nervous system (CNS) and the neural complications which result.9Lavi E Kolson DL Ulrich AM Fu L Gonzalez-Scarano F Chemokine receptors in the human brain and their relationship to HIV infection.J Neurovirol. 1998; 4: 301-311Crossref PubMed Scopus (105) Google Scholar, 10Gabuzda D He J Ohagen A Vallat A Chemokine receptors in HIV-1 infection of the central nervous system.Semin Immunol. 1998; 10: 203-213Crossref PubMed Scopus (70) Google Scholar Thus, it is critical to determine the expression and regulation of chemokines and their receptors in the CNS and how this is affected by HIV infection. Chemokine receptors are expressed constitutively in the CNS whereas chemokines are rarely detected in normal CNS but are highly expressed during a variety of CNS pathologies. We and others have demonstrated the expression of chemokines in the CNS in inflammatory pathologies including MIP-1α, MIP-1β, MCP-1, MCP-2, and MCP-311McManus CM Berman JW Brett FM Staunton H Farrell M Brosnan CF MCP-1, MCP-2, and MCP-3 expression in multiple sclerosis lesions: an immunohistochemical and in situ hybridization study.J Neuroimmunol. 1998; 86: 20-29Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 12Simpson JE Newcombe J Cuzner ML Woodroofe MN Expression of monocyte chemoattractant protein-1 and other beta chemokines by resident glia and inflammatory cells in multiple sclerosis.J Neuroimmunol. 1998; 84: 238-249Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 13Sasseville VG Smith MM Mackay CR Pauley DR Mansfield KG Ringler DJ Lackner AA Chemokine expression in simian immunodeficiency-induced AIDS encephalitis.Am J Pathol. 1996; 151: 1341-1351Google Scholar, 14Schmidtmayerova H Nottet HS Nuovo G Raabe T Flanagan CR Dubrovsky L Gendelman HE Cerami A Bukrinsky M Sherry B Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes.Proc Natl Acad Sci USA. 1996; 93: 700-704Crossref PubMed Scopus (263) Google Scholar and several recent reports demonstrate the expression of various chemokine receptors in the CNS.9Lavi E Kolson DL Ulrich AM Fu L Gonzalez-Scarano F Chemokine receptors in the human brain and their relationship to HIV infection.J Neurovirol. 1998; 4: 301-311Crossref PubMed Scopus (105) Google Scholar, 15Rottman JB Ganley KP Williams K Wu L Mackay CR Ringler DJ Cellular localization of the chemokine receptor CCR5.Am J Pathol. 1997; 151: 1341-1351PubMed Google Scholar, 16Vallat A-V Girolami UD He J Mhashilkar A Marasco W Shi B Gray F Bell J Keohane C Smith TW Gabuzda D Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brains of children with AIDS.Am J Pathol. 1998; 152: 167-178PubMed Google Scholar, 17Westmoreland SV Rottman JB Williams KC Lackner AA Sasseville VG Chemokine receptor expression on resident and inflammatory cells in the brains of macaques with simian immunodeficiency virus encephalitis.Am J Pathol. 1998; 152: 659-665PubMed Google Scholar, 18Xia M Qin S Wu L Mackay C Hyman BT Immunohistochemical study of the beta-chemokine receptors CCR3 and CCR5 and their ligands in normal and Alzheimer's disease brains.Am J Pathol. 1998; 153: 31-37Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar CXCR4 has been shown to be expressed on astrocytes, microglia, and neurons as has CCR5 in normal CNS (reviewed in 19Hesselgesser J Horuk R Chemokine and chemokine receptor expression in the central nervous system.J Neurovirol. 1999; 5: 13-26Crossref PubMed Scopus (249) Google Scholar). In this report, we analyze tissue sections from brains of pediatric acquired immune deficiency syndrome patients, with and without encephalitis, as well as aged-matched normal control tissue for the expression of the C-C chemokines, MIP-1α, MIP-1β, and MCP-1 and the chemokine receptors CCR2, CCR5, and CXCR4.Microglia have been shown to be the primary productively infected cell type of the CNS8Kolson DL Lavi E Gonzalez-Scarano F The effects of HIV in the CNS.Adv Virus Res. 1998; 50: 1-47Crossref PubMed Scopus (123) Google Scholar, 20Dickson DW Mattiace LA Kure K Hutchins K Lyman WD Brosnan CF Microglia in human disease, with an emphasis on acquired immune deficiency syndrome.Lab Invest. 1991; 64: 135-156PubMed Google Scholar whereas astrocyte infection, although reported, is controversial.21Nath A Hartloper V Furer M Fowke KR Infection of human fetal astrocytes with HIV-1: viral tropism and the role of cell to cell contact in viral transmission.J Neuropathol Exp Neurol. 1995; 54: 320-330Crossref PubMed Scopus (97) Google Scholar, 22Dewhurst S Sakai K Stevenson M Evinger-Hodges MJ Volsky DJ Persistent productive infection of human glial cells by HIV and by infectious molecular clones of HIV.J Virol. 1987; 61: 3774-3782PubMed Google Scholar Levels of virus in the CNS do not always correlate with neurological dysfunction and microglial activation is common in areas of the CNS where HIV antigen is not present.23Bossi P Dupin N Coutellier A Bricaire F Lubetzki C Katlama C Calvez V The level of human immunodeficiency virus (HIV) type 1 RNA in cerebrospinal fluid as a marker of HIV encephalitis.Clin Infect Dis. 1998; 26: 1072-1073Crossref PubMed Scopus (35) Google Scholar Thus, soluble factors released from HIV-infected cells may have effects on uninfected cells. Tat, an HIV transactivator protein, is secreted from HIV-infected cells24Jeang KT Tat structure and function.in: Meyers G Henderson LE Korber B Jeang KT Wain-Hobson S Human Retroviruses and AIDS. Los Alamos National Laboratory, Los Alamos1996: 3-18Google Scholar, 25Tardieu M Hery C Peudenier S Boespflug O Montagnier L Human immunodeficiency virus type 1-infected monocytic cells can destroy human neural cells after cell-to-cell adhesion.Ann Neurol. 1992; 32: 11-17Crossref PubMed Scopus (136) Google Scholar, 26Ensoli B Buonaguro L Barillari G Fiorelli V Gendelman R Morgan RA Wingfield P Gallo RC Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.J Virol. 1993; 67: 277-287Crossref PubMed Google Scholar by a leaderless pathway.27Cheng J Nath A Knudsen B Hochman S Geiger JD Ma M Magnuson DS Neuronal excitatory properties of human immunodeficiency virus type 1 Tat protein.Neuroscience. 1998; 82: 97-106Crossref PubMed Scopus (130) Google Scholar Little is known about the in vivo effects of this extracellular protein, particularly within the CNS. However, a recent report by Jones and colleagues28Jones M Olafson K Bigio MRD Peeling J Nath A Intraventricular injection of human immunodeficiency virus type 1 (HIV-1) tat protein causes inflammation, gliosis, apoptosis, and ventricular enlargement.J Neuropathol Exp Neurol. 1998; 57: 563-570Crossref PubMed Scopus (143) Google Scholar shows that intraventricular injection of Tat into male rats results in ventricular enlargement, apoptosis, and inflammation. Evidence for the expression of Tat within the CNS is reported29Wiley CA Baldwin M Achim CL Expression of HIV regulatory and structural mRNA in the central nervous system.AIDS. 1997; 10: 843-847Crossref Scopus (118) Google Scholar, 30Wesselingh S Levine B Fox RJ Choi S Griffin DE Intracerebral cytokine mRNA expression during fatal and nonfatal alphavirus encephalitis suggests a predominant type 2 T cell response.J Immunol. 1994; 152: 1289-1297PubMed Google Scholar and Tat has also been detected in the serum of patients infected with HIV.31Westendorp MO Frank R Ochsenbauer C Stricker K Dhein J Walczak H Debatin KM Krammer PH Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.Nature. 1995; 375: 497-500Crossref PubMed Scopus (910) Google Scholar There is a growing literature on the in vitro effects of Tat. Tat has been shown to mimic certain properties of C-C chemokines32Albini A Ferrini S Bernelli R Sforzini S Giunciuglio D Aluigi MG Proudfoot AE Alouani S Wells TN Mariani G Rabin RL Farber JM Noonan DM HIV-1 Tat protein mimicry of chemokines.Proc Natl Acad Sci USA. 1998; 95: 13153-13158Crossref PubMed Scopus (244) Google Scholar and can up-regulate CXCR4 on resting CD4+ T cells.33Secchiero P Zella D Capitani S Gallo RC Zani G Extracellular HIV-1 Tat protein up-regulates the expression of surface CXC-chemokine receptor 4 in resting CD4+ T cells.J Immunol. 1999; 162: 2427-2431PubMed Google Scholar With regards to the CNS, data indicates that Tat induces cytokine and adhesion molecule expression by brain microvascular endothelial cells as well as glial cells.34Chang HC Samaniego F Nair BC Buonaguro L Ensoli B HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region.AIDS. 1997; 11: 1421-1431Crossref PubMed Scopus (391) Google Scholar, 35Zidovetzki R Wang JL Chen P Jeyaseelan R Hofman F Human immunodeficiency virus tat protein induces interleukin 6 mRNA expression in human brain endothelial cells via protein kinase C dependent protein kinase pathways.AIDS Res Hum Retroviruses. 1998; 14: 825-833Crossref PubMed Scopus (76) Google Scholar, 36Sawaya BE Thatikunta P Denisova L Brady J Khalili K Amini S Regulation of TNF-alpha and TGF-beta1 gene transcription by HIV-1 tat in CNS cells.J Neuroimmunol. 1998; 87: 33-42Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar It has been reported to have potent neurotoxic effects27Cheng J Nath A Knudsen B Hochman S Geiger JD Ma M Magnuson DS Neuronal excitatory properties of human immunodeficiency virus type 1 Tat protein.Neuroscience. 1998; 82: 97-106Crossref PubMed Scopus (130) Google Scholar, 37Shi B Raina J Lorenzo A Busciglio J Gabuzda D Neuronal apoptosis induced by HIV-1 tat protein and TNF-alpha: potentiation of neurotoxicity mediated by oxidative stress and implications for HIV-1 dementia.J Neurovirol. 1998; 4: 281-290Crossref PubMed Scopus (174) Google Scholar and a recent report by Conant and colleagues38Conant K Garzino-Demo A Nath A McArthur JC Halliday W Power C Gallo RC Major EO Induction of monocyte chemoattractant protein-1 in HIV-1 tat stimulated astrocytes and elevation in AIDS dementia.Proc Natl Acad Sci USA. 1998; 95: 3117-3121Crossref PubMed Scopus (501) Google Scholar showed that Tat can induce MCP-1 in astrocytes. For this study, we analyzed the effects of the HIV protein, Tat, on chemokine and chemokine receptor expression in human fetal astrocytes and microglia.We have shown previously that astrocytes and microglia produce C-C chemokines in response to proinflammatory cytokines.39McManus CM Brosnan CF Berman JW Cytokine-induced MIP-1alpha and MIP-1beta expression in human fetal microglia.J Immunol. 1998; 160: 1449-1455PubMed Google Scholar Cytokines are major mediators of the inflammatory response serving to activate cells and mediate host responses. Chemokines are present in the local environment of the inflammatory response and studies indicate an essential role for chemokines in the establishment of this response because several studies have shown that blocking chemokine expression can ameliorate inflammatory disease.40Karpus WJ Lukacs NW McRae BL Strieter RM Kunkel SL Miller SD An important role for the chemokine macrophage inflammatory protein-1 alpha in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis.J Immunol. 1995; 155: 5003-5010PubMed Google Scholar, 41Karpus WJ Kennedy KJ MIP-1alpha and MCP-1 differentially regulate acute and relapsing autoimmune encephalomyelitis as well as Th1/Th2 lymphocyte differentiation.J Leukoc Biol. 1997; 62: 681-687PubMed Google Scholar Chronic neurological dysfunction can result in part from a continuing inflammatory response. How this response is perpetuated is not clearly understood. We determined whether chemokines could act in an autocrine fashion to induce their own expression, and thus, play a role not only in the inflammatory response but also in the perpetuation of this response. We analyzed the pattern of expression of chemokines and the C-C chemokine receptors, CCR1-CCR5, in response to chemokine treatment of human fetal astrocytes and microglia and show a novel autocrine function for chemokines.Materials and MethodsTissue Collection and PreparationImmunocytochemical studies were performed on brain tissue taken at autopsy. Seven pediatric patients with HIV encephalitis were studied along with nonencephalitic brains from two patients with HIV infection and three age-matched control brains (Table 1). Astroglioma tissue and lung tissue from patients with pneumonia were used as positive controls because these tissues are known to express high levels of chemokines.Table 1Immunocytochemical StudiesCaseHIV statusAge/sexBrain pathology1+1 y/FHIV encephalitis, HIV leukoencephalopathy2+6.5y/MHIV encephalitis, calcific vasculopathy3+6 y/MHIV encephalitis, multiple encephalomalacia4+9 wk/FHIV encephalitis, calcific vasculopathy5+2.5 y/MHIV encephalitis, HIV leukoencephalopathy, calcific vasculopathy6+14 wk/FFocal chronic meningitis7+5.5 mo/MMetabolic gliosis, calcific vasculopathy8−16 mo/FNormal brain9−1 y/MNormal brain10−2 y/MNormal brain Open table in a new tab AntibodiesPrimary antibodies for use in immunohistochemistry were obtained from the following sources: MCP-1, MIP-1α, and MIP-1β were kindly provided by Leukosite Inc. (Cambridge, MA). MCP-1 and MIP-1α are purified monoclonal antibodies used at a concentration of 2.8 μg/ml and MIP-1β was an ascites used at a dilution of 1:250. All antibodies were screened by Leukosite for reactivity to chemokines on paraffin-embedded tissues. CCR2, CCR5, and CXCR4 antibodies were kindly provided by Berlex Biosciences (San Francisco, CA) and used at a concentration of 5 μg/ml. Isotype-matched antibodies, IgG1, IgG2a, and IgG2b were purchased from Cappel (Durham, NC) and used as a negative control at a concentration of 2.8 μg/ml.ImmunohistochemistryParaffin-embedded tissue was dehydrated in graded alcohol baths and then deparaffinized in xylene. After rehydration the sections were quenched for 20 minutes in 0.8% hydrogen peroxide in methanol. Sections were then incubated in 2% normal horse serum (Vector Laboratories, Burlingame, CA) followed by an overnight incubation at 4°C in primary antibody. The sections were washed and incubated with a biotinylated secondary antibody (1:750; Vector Laboratories) followed by incubation in avidin-biotin-peroxidase complex (Vector Laboratories). The slides were developed with 3′3′-diaminobenzidene to give a brown reaction product (Sigma, St. Louis, MO) and then dehydrated and mounted with Cytoseal (VWR, Boston, MA).Immunoelution of TatProtein A agarose beads were pelleted and washed and purified rabbit anti-Tat (1:50 dilution) was added and incubated for 1 hour at room temperature. After washing, Tat was added at the treatment concentration for 1 hour at room temperature. After centrifugation, the supernatant was used in the enzyme-linked immunosorbent assay (ELISA. studies.Cell Culture and ReagentsHuman fetal CNS tissue (20 to 23 weeks) was obtained at the time of elective termination of intrauterine pregnancy from otherwise healthy females. Informed consent was obtained from all participants. This tissue was used as part of an ongoing research protocol that has been approved by the Albert Einstein College of Medicine Committee on Clinical Investigation and the City of New York Health and Hospitals Corporation. Microglia and astrocyte cultures were prepared as previously described.39McManus CM Brosnan CF Berman JW Cytokine-induced MIP-1alpha and MIP-1beta expression in human fetal microglia.J Immunol. 1998; 160: 1449-1455PubMed Google Scholar, 42Lee SC Liu W Brosnan CF Dickson DW Characterization of primary human fetal dissociated central nervous system cultures with an emphasis on microglia.Lab Invest. 1992; 67: 465-476PubMed Google Scholar Briefly, tissue was dissociated and incubated for 45 minutes at 37°C in 1× Hanks' balanced salt solution (Life Technologies, Inc., Grand Island, NY), 1× trypsin (Life Technologies, Inc.), and DNase 1 (Boehringer-Mannheim, Indianapolis IN). Tissue fragments were passed through 250- and 150-μm nylon mesh (Tetko, Inc., Briar Cliff Manor, NY). Cells were washed and resuspended in complete Dulbecco's modified Eagle's medium (25 mmol/L HEPES, 10% fetal calf serum, 1% nonessential amino acids, and 1. penicillin-streptomycin) and rewashed. Cells were seeded at 1.2 × 108 cells per 150-cm2 tissue-culture flask (Falcon; Becton Dickinson, Franklin Lakes, NJ) and cultured for 12 days. Microglial cells were then removed from the mixed culture by shaking for 30 minutes at 4°C and plated in complete Dulbecco's modified Eagle's medium at a concentration of 1 × 106 cells per 20-cm2 tissue-culture plate (Falcon). Cells were analyzed for the purity of the culture and shown to be ≥95% HAM56 (a microglial marker) positive. Astrocytes, the adherent population remaining in the 150-cm2 flask, were placed in RPMI 1640 medium with 10% fetal calf serum and 1. penicillin-streptomycin. These cells were then passaged several times and allowed to grow to confluency. The astrocyte cultures were ≥95. GFAP-positive (an astrocyte marker). Cells were treated with human recombinant MIP-1α, MIP-1β at 1 to 100 ng/ml (R&D Systems), MCP-1 at 1 to 100 ng/ml (Pharmingen, San Diego, CA), or HIV Tat protein at 1 to 100 ng/ml (prepared as previously described43Ma M Nath A Molecular determinants for cellular uptake of Tat protein of human immunodeficiency virus type 1 in brain cells.J Virol. 1997; 71: 2495-2499Crossref PubMed Google Scholar). Briefly, the recombinant Tat was prepared by expressing the tat gene encoding amino acids 1 to 72 (first exon) as a fusion protein in Escherichia coli DH5αf'1Q (Life Technologies, Inc.) and purifying it with a metal chelate affinity column. Tat was diluted with the following buffer before use: 50 mmol/L Tris, pH 8.0; 100 mmol/L NaCl; 1 mmol/L CaCl2; 0.5 mmol/L dithiothreitol. Endotoxin levels for all of the chemokines and the Tat were <1 ng/ml as tested by Limulus assay (BioWhitaker, Walkersville, MD). To accurately reflect donor variation each donor is reported as a separate symbol in all figures. Cell numbers obtained from each donor also varied due to tissue sample size and cell recovery, accounting for differences in numbers of untreated versus treated conditions.RNA Extraction and RNase Protection AssayTotal RNA was extracted from microglial cultures using Tri-Reagent (Molecular Research Center, Cincinnati, OH). Probe cocktails of C-C chemokines (panel CR5), C-C chemokine receptors (panel CK5), and C-X-C chemokine receptors (panel CR6) were obtained from Pharmingen. Ambion Maxiscript kit was used according to manufacturer's instruction for the generation of the RNA probes and the Ambion RPA II kit was used according to manufacturer's instructions for the analysis of mRNA expression. Samples were analyzed by 5% denaturing acrylamide gels followed by autoradiography (Fischer, Springfield, NJ). Densitometry was performed on multiple film exposures using densitometric software (NIH Shareware).Chemokine ELISASupernatants from microglial cell cultures were analyzed for chemokine proteins. Matched antibody pairs for MIP-1α and MIP-1β were purchased from R&D Systems (Minneapolis, MN) for use in sandwich ELISA. Antibody pairs for MCP-1 were purchased from Pharmingen. Ninety-six well plates were coated with chemokine antibody at a concentration of 4 μg/ml in 1× phosphate-buffered saline (PBS) at 4°C overnight. Plates were then washed with 1× PBS with 0.005. Tween-20 (Bio-Rad, Hercules, CA) and blocked with 1% bovine serum albumin in PBS. Samples were then added to the plate and a standard curve was generated using a series of dilutions from 2000 pg/ml to 31.25 pg/ml of the appropriate recombinant human chemokine (MIP-1α, MIP-1β, R&D Systems; MCP-1, Pharmingen). Samples and standards were allowed to incubate overnight at 4°C. Plates were then washed again and incubated for 1 hour at room temperature with specific biotinylated secondary antibody, washed, and incubated for 30 minutes in avidin-peroxidase (Sigma). The plates were washed, TMB (KPL, Gaithersburg, MD) substrate added for 5 minutes or less and the reaction stopped with 1 mol/L phosphoric acid. Absorbance was read at 450-nm wavelength on a microplate reader (Bio-Rad) within 30 minutes of stopping the reaction.Statistical AnalysesStatistical significance for microglia and astrocyte chemokine protein expression was determined using the Wilcoxon signed rank test. This test is nonparametric and as such analyzes each experimental group individually, allowing for an accurate measurement of variability in chemokine induction. This test does not generate error bars because the P value is not based on the means of pooled experiments. Individual points are represented in the graphs to illustrate the variability between experiments. Densitometric results of the RNase protection assays were analyzed using the student's paired t-test. Significance, with both analyses, was assigned for P ≤ 0.05 (StatView; Abacus Concepts, Berkeley, CA).ResultsImmunohistochemical Analysis of MIP-1α, MIP-1β, and MCP-1 ExpressionStaining of pediatric tissues showed that chemokines are highly expressed in the brains of children with HIV encephalitis but are not constitutively expressed in the brains of children with non-CNS-related pathologies. Figure 1A shows MIP-1α staining of a highly encephalitogenic case of HIV encephalitis where immunoreactivity is noted in the parenchyma by glial cells. Figure 1B, an age-matched control tissue with non-CNS-related pathologies, is negative for MIP-1α staining and is representative of staining of normal tissue with MIP-1β (data not shown). Figures 1C and 1D illustrate MIP-1β expression in HIV encephalitis particularly around blood vessels by glial cells and perivascular mononuclear cells (Figure 1D) and in the nonvessel-associated white matter (Figure 1C). Staining for MIP-1β is intense on astrocytes as well as microglia and, similar to MIP-1α, staining is seen on mononuclear cells infiltrating the CNS. MCP-1 is expressed by astrocytes, microglia, and mononuclear cells as well as on the endothelium (Figure 1, E and F). Figure 1G illustrates the lack of MCP-1 immunoreactivity in age-matched control tissue. Figure 1H is an isotype-specific negative control reagent that is nonreactive in the tissues analyzed and is representative of all isotype-matched controls. A summary of the immunohistochemical results is shown in Table 2.Table 2Immunoreactivity of Chemokines and Chemokine Receptors in HIV EncephalitisAstrocytesMicrogliaEndotheliumPBMCsNeuronsChemokinesMIP-1α++++++−++−/+MIP-1β++++++−++−/+MCP-1++++++++++Chemokine receptorsCCR2++−+−CCR5++++−+++CXCR4+++++++++++Immunoreactivity was determined on an arbitrary scale; −, no reactivity; +++, extensive reactivity. Open table in a new tab Immunohistochemical Analysis of CCR2, CCR5, and CXCR4 ExpressionAnalyses of chemokine receptor expression in pediatric HIV encephalitis demonstrated that the expression of these receptors is not always restricted to disease states, in that some of the receptors were also expressed in normal brains. CCR2 was not detected in the parenchyma of normal brains (Figure 2B), although it was expressed on glial cells and peripheral mononuclear cells within vessels in HIV encephalitis (Figure 2A). This differs from what was seen with CCR5 and CXCR4 which were present in both normal (Figure 2, D and F) and HIV encephalitogenic brains (Figure 2, C and E). This correlates with recent data showing immunoreactivity for CCR5 by glial cells and mononuclear cells with staining being more intense in the encephalitic cases.16Vallat A-V Girolami UD He J Mhashilkar A Marasco W Shi B Gray F Bell J Keohane C Smith TW Gabuzda D Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brains of children with AIDS.Am J Pathol. 1998; 152: 167-178PubMed Google Scholar Anti-CXCR4 stained neurons as well as glial cells and mononuclear cells in encephalitogenic brains and in normal brains (Figure 2, E and F), again with more intense staining noted in the encephalitic brains which confirms previous data by Vallat et al.16Vallat A-V Girolami UD He J Mhashilkar A Marasco W Shi B Gray F Bell J Keohane C Smith TW Gabuzda D Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brains of children with AIDS.Am J Pathol. 1998; 152: 167-178PubMed Google Scholar Most of the sections analyzed were from the cortex and cerebellum. There were variations in the intensity of staining observed with both chemokines and chemokine receptors between different individuals, and the areas of tissue chosen for illustration are most representative of the intensity of staining seen.Figure 2Immunohistochemical analysis of chemokine re" @default.
• W87068116 created "2016-06-24" @default.
• W87068116 creator A5000097172 @default.
• W87068116 creator A5006246174 @default.
• W87068116 creator A5026869884 @default.
• W87068116 creator A5037794520 @default.
• W87068116 creator A5047208383 @default.
• W87068116 creator A5057650635 @default.
• W87068116 creator A5061541471 @default.
• W87068116 date "2000-04-01" @default.
• W87068116 modified "2023-10-16" @default.
• W87068116 title "Chemokine and Chemokine-Receptor Expression in Human Glial Elements" @default.
• W87068116 cites W1500908560 @default.
• W87068116 cites W1502746319 @default.
• W87068116 cites W1524845305 @default.
• W87068116 cites W1546946747 @default.
• W87068116 cites W1548598731 @default.
• W87068116 cites W1560316450 @default.
• W87068116 cites W1604626968 @default.
• W87068116 cites W1620558271 @default.
• W87068116 cites W1652237838 @default.
• W87068116 cites W1904940664 @default.
• W87068116 cites W1906710068 @default.
• W87068116 cites W1921100729 @default.
• W87068116 cites W1965019797 @default.
• W87068116 cites W1966700067 @default.
• W87068116 cites W1967681223 @default.
• W87068116 cites W1971642548 @default.
• W87068116 cites W1971864431 @default.
• W87068116 cites W1975803523 @default.
• W87068116 cites W1977473578 @default.
• W87068116 cites W1981644527 @default.
• W87068116 cites W1984811742 @default.
• W87068116 cites W1989035987 @default.
• W87068116 cites W1989584276 @default.
• W87068116 cites W1996183144 @default.
• W87068116 cites W1997653290 @default.
• W87068116 cites W2003060818 @default.
• W87068116 cites W2005659566 @default.
• W87068116 cites W2006733828 @default.
• W87068116 cites W2014964914 @default.
• W87068116 cites W2027580727 @default.
• W87068116 cites W2030055862 @default.
• W87068116 cites W2031196287 @default.
• W87068116 cites W2035134338 @default.
• W87068116 cites W2038418972 @default.
• W87068116 cites W2039683993 @default.
• W87068116 cites W2049163289 @default.
• W87068116 cites W2057379124 @default.
• W87068116 cites W2059726200 @default.
• W87068116 cites W2062200627 @default.
• W87068116 cites W2075110274 @default.
• W87068116 cites W2096550955 @default.
• W87068116 cites W2108499384 @default.
• W87068116 cites W2109098861 @default.
• W87068116 cites W2120658132 @default.
• W87068116 cites W2121356335 @default.
• W87068116 cites W2126597682 @default.
• W87068116 cites W2137334328 @default.
• W87068116 cites W2137800254 @default.
• W87068116 cites W2149787553 @default.
• W87068116 cites W2170991170 @default.
• W87068116 cites W2412971245 @default.
• W87068116 cites W2419362345 @default.
• W87068116 cites W4211264883 @default.
• W87068116 cites W4313333659 @default.
• W87068116 doi "https://doi.org/10.1016/s0002-9440(10)65013-4" @default.
• W87068116 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1876886" @default.
• W87068116 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10751368" @default.
• W87068116 hasPublicationYear "2000" @default.
• W87068116 type Work @default.
• W87068116 sameAs 87068116 @default.
• W87068116 citedByCount "163" @default.
• W87068116 countsByYear W870681162012 @default.
• W87068116 countsByYear W870681162013 @default.
• W87068116 countsByYear W870681162014 @default.
• W87068116 countsByYear W870681162015 @default.
• W87068116 countsByYear W870681162016 @default.
• W87068116 countsByYear W870681162017 @default.
• W87068116 countsByYear W870681162018 @default.
• W87068116 countsByYear W870681162019 @default.
• W87068116 countsByYear W870681162020 @default.
• W87068116 countsByYear W870681162021 @default.
• W87068116 countsByYear W870681162022 @default.
• W87068116 crossrefType "journal-article" @default.
• W87068116 hasAuthorship W87068116A5000097172 @default.
• W87068116 hasAuthorship W87068116A5006246174 @default.
• W87068116 hasAuthorship W87068116A5026869884 @default.
• W87068116 hasAuthorship W87068116A5037794520 @default.
• W87068116 hasAuthorship W87068116A5047208383 @default.
• W87068116 hasAuthorship W87068116A5057650635 @default.
• W87068116 hasAuthorship W87068116A5061541471 @default.
• W87068116 hasBestOaLocation W870681161 @default.
• W87068116 hasConcept C12823836 @default.
• W87068116 hasConcept C128879288 @default.
• W87068116 hasConcept C129013479 @default.
• W87068116 hasConcept C13373296 @default.
• W87068116 hasConcept C179661763 @default.

• next