UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
Immune responses to tuberculosis
Juffermans, N.P.
Publication date
2000
Link to publication
Citation for published version (APA):
Juffermans, N. P. (2000). Immune responses to tuberculosis. Thela Thesis.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.
Patientss with active tuberculosis have an increased expression of
HIVV coreceptors CXCR4 and CCR5 on CD4
+T cells
Nicolee P. Juffermans( ' ', Peter Speelman', Annelies Verbon' '°, Jan Veenstra", Cornellss Jie*, Sander J.H. van Deventer*, Tom van der Poll'V)
Fromm the Laboratory of Experimental Internal Medicine, the Department of Internal Medicine,, Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medicall Centre, University of Amsterdam and the ^Department of Internal Medicine
andd Pulmonary Care, Sint Lucas Hospital, Amsterdam, the Netherlands.
ChapterChapter 11
Abstract t
Replicationn of HTV is enhanced in HIV infected patients with active tuberculosis (TB),, returning to baseline after tuberculostatic treatment. Chemokine receptors CXCR44 and CCR5 can act as HIV coreceptors. We hypothesized that TB increases thee HIV load through upregulation of CXCR4 and CCR5. Expression of HIV-coreceptorss CXCR4 and CCR5 was found elevated on CD4+ T cells in patients with TB,, and after in vitro stimulation with mycobacterial lipoarabinomannan. These data suggestt that the increase in HIV viremia during tuberculosis may occur through upregulationn of CXCR4 and CCR5 on CD4+ T cells, thereby accelerating HIV disease. .
Introduction n
Inn up to 50-67% of HIV patients, tuberculosis (TB) is the AIDS-defining diagnosis. Concurrentt infection with TB results in an enhanced susceptibility of immune cells forr HIV infection, facilitating HIV entry and replication [1, 2]. In vitro, monocytes of patientss with TB are more susceptible to HIV-infection [1]. Moreover, viral replicationn is increased in HIV-infected patients who develop active TB, returning to baselinee after treatment [2]. This finding is of clinical relevance since patients with TBB have an accelerated course of their HIV-infection. The chemokine receptors CXCR44 and CCR5 act as coreceptors for HIV entry into CD4+ T cells [3]. HIV coreceptorr expression correlates with enhanced HIV entry into cells and HIV replicationn [4, 5]. We hypothesized that TB stimulates HIV coreceptor expression, therebyy enhancing HIV entry into immune cells and HIV replication.
Too investigate HIV coreceptor expression during TB, we measured CXCR4 and CCR55 by FACSanalysis after in vitro stimulation of whole blood with lipoarabinomannann (LAM), a cell wall component of Mycobacterium tuberculosis andd in patients with TB.
Methods s
InIn vitro stimulation. Blood was collected from six healthy subjects using a sterile
collectingg system consisting of a butterfly needle connected to a syringe (Becton Dickinson,, Mountain View, CA) and incubated at 37°C for 8 hours. Anticoagulation wass obtained using heparin (Leo Pharmaceutical Products, Weesp, the Netherlands; finall concentration 10 U/ml blood). Whole blood was added to sterile polypropylene tubess and diluted 1:1 with RPMI 1640 (Bio Whittaker,Verviers, Belgium), to which LAMM (mannose-capped, isolated and prepared from M. tuberculosis strain H37Rv), kindlyy provided by J.T. Belisle (Colorado State University, Fort Collins, CO, under Nationall Institutes of Health Contract NO1-A1-75320) was added at a concentration off 1 u,g/ml and stimulated for 8 hours at 37°C, after which fluorescence-activated cell sorterr (FACS) analysis was performed.
PatientsPatients and controls. Blood was obtained from eight patients with active, culture
provenn TB attending the Academic Medical Center (n=5), the Sint Lucas Hospital (n=2)) and the Municipal Health Center (n=l) in Amsterdam, the Netherlands, with a meann ) age of 31.9 2 yrs). Three patients with TB were HTV-seropositive,
ChapterChapter 11
determined.. Of the patients, 4 had pulmonary tuberculosis and 4 had extrapulmonary tuberculosis.. On each day a patient was analyzed, blood was also drawn from 8 healthyy HIV-seronegative controls 0 yrs). After collection, blood was immediatelyy prepared for FACS analysis.
FlowFlow cytometry. Blood was prepared for FACS analysis as follows. Erythrocytes weree lysed with bicarbonate buffered ammonium chloride solution (pH 7.4). Leukocytess were recovered after centrifugation at 1450 rpm for 5 minutes and counted.. 1 x 106 cells were resuspended in phosphate-buffered saline containing EDTAA lOOmM, sodium azide 0.1% and bovine serum albumin 5% (cPBS) and placedd on ice. Triple staining was obtained by incubation for 1 hour with direct labeledd antibodies CD3-PE, CD4-Cy (both from Coulter Immunotech) and either CXCR4-FITCC or CCR5-FITC (R&D Systems, Abingdon, United Kingdom). Blood wass also incubated with FITC-labeled CD25 (CLB) and PE-labeled CD69 (Becton & Dickinson).. Nonspecific staining was controlled for by incubation of cells with FITC-labelledd mouse IgG2 (Coulter Immunotech, Marseille, France). Cells were thenn washed twice in ice cold cPBS and resuspended for flow cytofluorometric analysiss (Calibrite; Becton Dickinson Immunocytometry Systems, San Jose, CA). At leastt 10,000 lymphocytes were counted. Data on the number of positive cells were obtainedd by setting a quadrant marker for nonspecific staining.
StatisticalStatistical analysis. Results are expressed as mean SE unless stated otherwise. Data weree analyzed using the Wilcoxon test. P<0.05 was considered statistically significant. .
Figuree 1.
Expressionn of CXCR4 and CCR5 onn CD4* T cells after whole bloodd incubation with Lipoarabinnomannann (LAM, 1 ug/ml)) for 8 hours. *P<0.05 versuss incubation with RPMI medium. .
LAM M RPMI I
CXCR4 4 CCR5 5
Results s
Whenn compared to incubation with medium alone, LAM induced an upregulation of thee fraction of CD4+ T cells positive for CXCR4 (39.0 4.8 vs. 24.7 4.1 %) and forr CCR5 (27.5 5.5 vs 4.3 1.7 %, P<0.05 for both, Figure 1) after stimulation of wholee blood in vitro.
P=0.002 2 So So So o 100j j 75-- 50-- 25--o0o o P=0.002 2 o o o o o o CXCR4 4 CCR5 5 TB oo TB HIV+ oo control Figuree 2. Expressionn of CXCR4 and CCR55 on circulating CD4+ T cellss of 8 patients with active tuberculosiss (TB) and 8 healthy controls. .
Havingg established that part of the cell wall of M. tuberculosis can upregulate HIV coreceptorr expression, we next determined CXCR4 and CCR5 in patients with active TB.. The percentage of circulating CD4+ T cells and CD8+ T cells in patients did not differr from controls (CD4: 41.3+4.7 versus 6 %; CD8: 3 versus
55 %, NS). The fraction of circulating CD4+ T cells positive for CXCR4 and CCR55 was higher in TB patients than in healthy controls (Figure 2, P<0.005). The fractionn of circulating CD4+ T cells expressing lymphocyte activation markers CD25
orr CD69 did not differ between patients and controls (CD25: 19.8 (1.0-44.3) versus 25.33 (2.7-54.4), NS; CD69: 9.2 (0.4-74.6) versus 7.2 (0.3-54.4), NS), suggesting that thee observed upregulation is due to specific receptor stimulation by antigens and not duee to an activated state of lymphocytes during TB.
Discussion n
Thee relation of CXCR4 and CCR5 with HIV disease has been clearly demonstrated [3],, making knowledge of FIIV coreceptor expression during concurrent infection a clinicallyy important issue. This study is the first to report elevated CXCR4 and CCR5 expressionn in patients with TB and after in vitro stimulation with an antigen derived fromm M. tuberculosis. Previously, it has been found that LAM can stimulate HIV
ChapterChapter 11
mayy occur through upregulation of CXCR4 and CCR5 on CD4+ T cells in HIV patients,, at least in part mediated by LAM, thereby accelerating HIV disease.
HTV-seropositivee patients have increased CCR5 expression compared to HIV-seronegativee controls [7]. In accordance, the two TB patients who were HIV-positive hadd the highest fraction of CD4+ T cells positive for CCR5. In contrast, CXCR4 has beenn found at low levels in HTV-seropositive patients [7]. We found elevated expressionn of CXCR4 in TB patients, with the two HIV-seropositive patients expressingg high and intermediate levels. The relation of CXCR4 and HIV disease is lesss clear then for CCR5 [3], and needs further investigation. In the two HIV-seronegativee TB patients, expression of CXCR4 and CCR5 showed little overlap withh healthy controls, suggesting that the elevated levels of CXCR4 and CCR5 duringg TB is not attributable to HIV alone.
HTVV coreceptors are considered goals for HIV therapy. This study contributes to the ideaa that blocking CXCR4 and CCR5 may slow down disease progression during concurrentt infection [3].
References s
1.. Toossi Z, Sierra-Madero JG, Blinkhorn RA, Mettler MA, Rich EA. Enhanced susceptibility of bloodd monocytes from patients with pulmonary tuberculosis to productive infection with human immunodeficiencyy virus type 1. J Exp Med 1993;177:1511-6.
2.. Goletti D, Weissman D, Jackson RW, Graham NM, Vlahov D, Klein RS, Munsiff SS, Ortona L, Caudaa R, Fauci AS. Effect of Mycobacterium tuberculosis on HIV replication. Role of immune activation.. J Immunol 1996;157:1271-8.
3.. Berger EA, Murphy, Philip M., Farber, Joshua M. Chemokine receptors as HIV-1 coreceptors: Roless in Viral Entry, Tropism, and Disease. Annu. Rev. immunol. 1999;17:657-700.
4.. Dolei A, Biolchini A, Serra C, Curreli S, Gomes E, Dianzani F. Increased replication of T-cell-tropicc HIV strains and CXC-chemokine receptor-4 induction in T cells treated with macrophage inflammatoryy protein (MlP)-lalpha, MlP-lbeta and RANTES beta-chemokines. AIDS 1998;12:183-90. .
5.. Wahl SM, Greenwell-Wild T, Peng G, Hale-Donze H, Doherty TM, Mizel D, Orenstein JM. Mycobacteriumm avium complex augments macrophage HIV-1 production and increases CCR5 expression.. Proc Natl Acad Sci USA 1998;95:12574-9.
6.. Peterson PK, Gekker G, Bornemann M, Chatterjee D, Chao CC. Thalidomide inhibits lipoarabinomannan-inducedd upregulation of human immunodeficiency virus expression. Antimicrobb Agents Chemo 1995;39:2807-9.
