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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.
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Expressionn of urokinase plasminogen activator receptor
andd CDllb during tuberculosis
Nicolee P. Juffermans(l' ', Pascale E.P. Dekkers1, Annelies Verbon(1'2), Peter Speelman2,, Sander J.H. van Deventer',Tom van der Poll(1'2)
Fromm the Laboratory of Experimental Internal Medicine, the ^Department of Internal Medicine,, Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medicall Center, University of Amsterdam, Amsterdam, the Netherlands
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Abstract t
Thee receptor for urokinase plasminogen activator (uPAR) is important for leukocyte migration.. The capacity of uPAR to function as an adhesion receptor is facilitated by complexx formation with complement receptor 3 (CR3), which is composed of CDllbb and CD18. Knowledge of regulation of leukocyte migration during tuberculosiss (TB) is limited. Therefore, we determined the expression of uPAR and CDllbb on circulating leukocytes of 8 patients with TB and 8 healthy controls. Expressionn of uPAR and CDllb was higher on monocytes in patients with TB than inn controls, whereas granulocytes showed non-significant increases. In vitro, lipoarabinnomannann (LAM), a cell wall component of M. tuberculosis and lipopolysaccharidee (LPS) from E. coli shared the ability to enhance uPAR and CDllbb on monocytes and granulocytes. Furthermore, intravenous administration of LPSS to 8 healthy humans resulted in an increase of uPAR and CDllb on monocytes andd granulocytes. We conclude that uPAR and CDllb are concurrently upregulated onn monocytes during TB, which may be at least in part mediated by mycobacterial LAM. .
Introduction n
Bothh mononuclear and polymorphonuclear cells are implicated in the host response too M. tuberculosis. The process of migration of leukocytes from the vascular space intoo inflamed lung tissue involves rolling of cells along the vessel wall, followed by firmm attachment to the endothelium and transmigration [1]. In this process, the P2 integrinn C D l l b , which together with CD 18 forms complement receptor 3 (CR3), mediatess vascular cell attachment. The receptor for urokinase plasminogen activator (uPAR,, CD87) can act as an adhesion receptor, and in vitro chemotaxis of monocytes andd granulocytes at least in part depends on the expression of uPAR [2-4]. uPAR is a glycosylphosphatidylinositol-linkedd receptor without a transmembrane domain, thereforee lacking a direct link to the cell interior. However, uPAR can form complexess with CR3 on monocytes [3, 5] and granulocytes [6, 7], thus resulting in signall transduction. Indeed, complex formation with uPAR facilitates the adhesive functionn of C D l l b on monocytes, since blocking anti-uPAR antibodies suppress C D l l bb dependent adhesion of monocytes [8]. Disruption of the uPAR-CR3 interactionn also inhibits granulocyte migration in vitro [9]. Taken together, the ability off uPAR to interact with CR3 seems important for adhesion and migration of monocytess and granulocytes.
Knowledgee of the regulation of cellular uPAR expression in relation with the surface expressionn of C D l l b in vivo is limited. Recently, we reported that a bolus intravenouss injection of lipopolysaccharide (LPS) into healthy humans was associatedd with an upregulation of monocyte uPAR expression, while the effect on granulocytee uPAR expression was inconsistent [10]. Thusfar, the expression of the uPAR/CR33 complex during infections in vivo has not been studied. Therefore, we foundd it of interest to measure cellular uPAR and CD1 lb expression in patients with tuberculosiss (TB). In addition, the capacity of lipoarabinnomannan (LAM, derived fromm M. tuberculosis) to influence cellular uPAR and C D l l b expression was comparedd with the effect of LPS, and the model of human experimental endotoxemia wass used to further study the kinetics of uPAR and C D l l b expression during inflammation. .
Methods s
PatientsPatients and controls. EDTA blood was obtained from 8 patients with culture proven
TB.. The mean age of TB patients was 32 4 yrs and did not differ from the 8 healthyy controls (29 2 yrs). Of the patients, 4 had pulmonary TB and 4 had
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extrapulmonaryy TB. At the same day a patient was analyzed, blood was also obtained fromm a healthy control and prepared for fluorescence-activated cell sorter (FACS) analysiss (Calibrite; Becton Dickinson Immunocytometry Systems, San Jose, CA).
WholeWhole blood stimulation. Blood was collected aseptically from six healthy subjects
andd diluted 1:1 with RPMI 1640 (Bio Whittaker,Verviers, Belgium) to which LAM (11 u,g/ml, prepared from M. tuberculosis strain H37Rv, provided by J.T. Belisle (Fort Collins,, CO, National Institutes of Health Contract NOl-A 1-75320) or LPS (10 ng/ml,, from Escherichia coli serotype 0111: B4; Sigma, St Louis, MO) was added, andd incubated at 37° C for 6 hours.
HumanHuman experimental endotoxemia. Eight healthy male subjects, age 24 2 years, weree admitted to the clinical research unit of the Academic Medical Center, after documentationn of good health. The participants did not smoke, used no medication andd had no febrile illness within two weeks prior to start of the study. The study was approvedd by the institutional research and ethics committees and written informed consentt was obtained. All volunteers received a bolus intravenous injection of LPS (fromm Escherichia coli, lot G, U.S. Pharmacopeial Convention, Rockville, MD) at a dosee of 4 ng/kg body weight. Venous blood samples were obtained directly before andd 6 hours after injection of LPS. The timing of blood sampling was based on our previouss study in which we determined the effect of intravenous LPS on cellular uPARR expression in humans in vivo [10].
FACSFACS analysis. Erythrocytes were lysed with bicarbonate buffered ammonium
chloridee solution (pH 7.4). After centrifugation, 1 x 106 leukocytes were resuspended inn PBS containing EDTA lOOmM, sodium azide 0.1% and bovine serum albumin 5%.. Blood of patients was incubated with FITC-labeled mouse anti-human uPAR mAbb (clone VIM-5; Instruchemie, Hilversum, the Netherlands) or with mouse anti-humann CD lib (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service,, CLB), Amsterdam, the Netherlands) followed by a FITC-labeled F(ab')2
goat-anti-mousee antibody (Zymed, San Francisco, CA). A murine isotype matched antibodyy was used to control for aspecific staining (IgGl, Becton, Dickinson & Co, Rutherford,, NJ). Blood from the in vitro experiments and blood from the volunteers receivingg LPS was incubated with PE-labeled mouse anti-human CDllb or mouse anti-humann uPAR (both from Pharmingen, San Diego, CA). Aspecific staining was controlledd for by incubation of cells with PE-labelled mouse IgGl (Coulter Immunotech,, Marseille, France). Data are presented as the difference between mean celll fluorescence (MCF) intensities of specifically and nonspecifically stained cells.
StatisticalStatistical analysis. Values are given as means SEM unless stated otherwise. Data weree analyzed using Wilcoxon test. P<0.05 was considered statistically significant.
Results s
ExpressionExpression of cellular uPAR and CD1 lb in patients with tuberculosis. Patients with
activee TB had increased expression of both uPAR and CD1 lb on the cell surface of circulatingg monocytes compared to healthy controls (P<0.05, Figure 1). Although granulocytee uPAR and granulocyte C D l l b tended to be higher in patients than in controls,, the differences did not reach statistical significance.
P<0.05 5 NS S a. a. << 400 a. a. 1 1 --0 --0 Oo o oo o o o 1 1 0 0 0 0 o o * * ^^ 2000 a a o o LL L O O ££ 1000-P<0.05 5 monocytes s NS S TB B control l granulocytes s Figuree 1.
Expressionn of monocyte and granulocytee uPAR and C D l l b inn 8 patients with tuberculosis andd 8 healthy controls. Data are expressedd as difference between specificc and nonspecific mean celll fluorescence (MCF). Horizontall lines represent medians.. NS = non significant.
ExpressionExpression of cellular uPAR and CDllb after in vitro stimulation with {mycobacterial{mycobacterial stimuli. Next we determined whether LAM, a cell wall component
off M. tuberculosis, can influence uPAR or C D l l b expression. For this purpose we incubatedd whole blood with LAM for 6 hours. LAM significantly enhanced expressionn of uPAR and C D l l b on both monocytes and granulocytes, although granulocytee upregulation was less prominent. Stimulation of whole blood with E.
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monocytes s granulocytes s
Figuree 2. Expression of monocyte and granulocyte uPAR and CD1 lb after whole blood stimulation
withh LPS (10 ng/ml) or LAM (1 u.g/ml) for 6 hours. Data are expressed as mean ( SE) difference betweenn specific and nonspecific mean cell fluorescence (MCF) of 6 donors. *P<0.05 for difference withh incubation of whole blood with medium (RPMI) alone.
ExpressionExpression of cellular uPAR and CDllb in experimental endotoxemia. Having
establishedd that LPS can upregulate cellular uPAR and C D l l b , we next used the humann experimental endotoxemia model to study uPAR and C D l l b expression duringg inflammation in vivo. Administration of LPS resulted in a strong upregulation off monocyte uPAR and C D l l b expression (both P<0.05), and a more modest increasee in granulocyte uPAR and C D l l b expression at 6 hours post injection (P<0.05,, Figure 3).
Discussion n
uPARR has been implicated as an important mediator of leukocyte trafficking. The abilityy of uPAR to function as an adhesion receptor depends on an association with CR33 (CD1 lb/CD 18) [2, 7, 8]. Here, we demonstrate an increase in the expression of uPARR and C D l l b on circulating monocytes of patients with TB, while granulocyte uPARR and C D l l b was only modestly affected. In whole blood, mycobacterial LAM ass well as LPS induced enhanced uPAR and C D l l b expression on monocytes and granulocytes.. Also, in human experimental endotoxemia, uPAR and C D l l b were increasedd on both monocytes and granulocytes. These data indicate that uPAR and C D l l bb are concurrently upregulated on monocytes, and to a lesser extent on
granulocytess during TB, at least in part mediated by the LAM component of the mycobacteriall cell wall. In this respect, the effect of LAM resembled that of LPS.
< < £200 0 £ £ * *
i i
Q Q O O £ £Figuree 3. Expression of monocyte
andd granulocyte uPAR and C D l l b afterr intravenous injection of LPS (44 ng/kg) into 8 subjects. Data are expressedd as mean ( SE) differencee between specific and nonspecificc mean cell fluorescence (MCF).. *P<0.05 compared to t=0 (beforee injection).
monocytess granulocytes
Thee host response to TB involves activation of mononuclear cells which in turn activatee CD4+ T cells, eventually resulting in the intracellular killing of tubercle bacillii [11], Mice injected with LPS showed increased levels of uPAR mRNA in theirr lung tissue, indicating a role for uPAR in the pulmonary compartment during infectionn [12]. Also, CDllb expression on alveolar macrophages of patients with TB iss increased compared to normal subjects [13], and CDllb/CD18 expression was associatedd with the capacity of macrophages to bind M. tuberculosis [14]. We found increasedd expression of uPAR and CDllb on monocytes but not on granulocytes in bloodd of patients with TB. Mycobacterial LAM upregulated uPAR and CDllb on monocytess and to a lesser extent on granulocytes. In accordance, uPAR mRNA was increasedd in macrophages, but not in granulocytes of mice injected with LPS [12]. Previously,, we found an upregulation of uPAR on monocytes of healthy humans injectedd with LPS, whereas the increase in uPAR expression on granulocytes was inconsistentt [10]. An explanation may be that other staining antibodies were used and/orr the slightly smaller study population in the earlier study. In this previous report,, also heat-killed S. aureus and staphylococcal enterotoxin B, a superantigen fromm S. aureus, upregulated monocyte uPAR in vitro [10]. Taken together, the data suggestt that M. tuberculosis and LAM may influence the function of monocytes by
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stimulatingg the surface expression of the adhesion mediating receptors uPAR and CDllb.. This effect is shared with both gram-positive and gram-negative bacterial stimuli.. Furthermore, our results suggest that monocytes are more sensitive than granulocytess in terms of uPAR upregulation upon stimulation.
References s
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