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Immune responses to tuberculosis
Juffermans, N.P.
Publication date
2000
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Citation for published version (APA):
Juffermans, N. P. (2000). Immune responses to tuberculosis. Thela Thesis.
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Serumm Concentrations of Lipopolysaccharide Activity-Modulating
Proteinss during Tuberculosis
Nicolee P. Juffermans(1,2), Annelies Verbon2, Sander J.H. van Deventer', Henk van Deutekomm , Peter Speelman2 Tom van der Poll"'2>
Fromm the Laboratory of Experimental Internal Medicine, the department of Internal Medicine,, Division of Infectious Diseases, Tropical Medicine and AEDS, Academic
Medicall Center, University of Amsterdam, Amsterdam, and the department of Tuberculosis,, Municipal Health Service, Amsterdam, the Netherlands
ChapterChapter 6
Abstract t
Lipopolysaccharidee (LPS) is the principal stimulator of host defense against gram-negativee bacteria. LPS binding protein (LBP), bactericidal/permeability increasing proteinn (BPI) and soluble CD14 (sCD14) bind LPS and regulate its toxicity. Lipoarabinomannann (LAM), a cell wall component of Mycobacterium tuberculosis, resembless LPS with respect to induction of inflammatory responses through recognitionn by LBP and sCD14. In this study LBP, BPI and sCD14 were measured in serumm of 124 patients with tuberculosis (TB) in various stages of disease, in persons whoo had been in close contact with patients with contagious pulmonary TB and in healthyy controls. Levels of these LPS-toxicity regulating proteins were elevated in patientss with active TB compared to contacts and controls, and declined dunng treatment.. The levels of LBP and sCDI4 were higher in patients with fever and anorexia.. LPS-regulating proteins may play a role in host defense during TB, presumablyy through interaction with LAM.
Introduction n
Itt has been estimated that one third of the world population is infected with
MycobacteriumMycobacterium tuberculosis. In industrialized countries the number of tuberculosis (TB)) cases has failed to decline [1]. Host defense mechanisms during TB and
pathwayss by which M. tuberculosis induces inflammatory responses are incompletelyy understood.
Lipopolysaccharidee (LPS) is the principal stimulator of host defense against gram-negativee bacteria. The biological availability of LPS is regulated by a number of serumm proteins, including LPS binding protein (LBP), bactericidal/permeability increasingg protein (BPI) and soluble CD14 (sCD14) [2]. LBP facilitates the binding off LPS to CD14, a glycoprotein expressed on monocytes and neutrophils that is essentiall for the induction of an inflammatory response to LPS. BPI is a protein secretedd by the azurophilic granules of neutrophils that binds and neutralizes LPS. LPSS activity can be further regulated by sCD14, the extracellular domain of cell-boundd CD 14, which can either enable LPS to activate cell types that lack membrane CD144 or inhibit LPS effects on CD14 expressing cell types by competition for the bindingg of LPS with cell-associated CD 14 [2].
Lipoarabinomannamm (LAM) is a lipid glycoprotein cell wall component of M.
tuberculosistuberculosis that has been implicated as a major factor in the induction of cytokine releasee during TB [3, 4]. LAM shares many physiochemical properties with LPS and
utilizess LBP, cell-associated CD14 and sCD14 in a similar way as LPS to exert inflammatoryy effects on cells [3-6]. Hence, it is conceivable that serum proteins involvedd in the regulation of LPS activity, also play a role in the regulation of the inflammatoryy response during TB by interference with the bioavailability of LAM. Therefore,, in the present study we determined serum concentrations of LBP, BPI and sCD144 in patients with TB before, during and after antituberculous treatment. Methods s
PatientPatient groups. Patient groups have been described elsewhere [7]. Sera were obtainedd from 82 patients with active, culture proven TB. 32% of these patients was female.. Mean age (range) was 35 years (15-86). Of these patients, 46 had pulmonary TBB and 36 had extrapulmonary TB. Extrapulmonary sites included lymph nodes (n=8),, pleura (n=12), bone and joints (n=6), soft tissue (n=2), meninges (n=3), and gastrointestinall tract (n=2). In 3 patients disease was disseminated. Sera were also obtainedd from 15 patients with TB who had received therapy for at least two weeks, butt had not yet completed therapy at the time of blood sampling, from 16 patients
ChapterChapter 6
whoo had completed therapy at least one month and not more than one year before bloodd sampling, and from 11 patients who had completed therapy at least one year andd not more than two years before blood sampling. Since analysis of the latter two groupss revealed no differences (data not shown), their results were combined. Of thesee 124 patients, 66 attended the Academic Medical Center and 58 the Municipal Healthh Service in Amsterdam, the Netherlands. There was no significant difference inn ethnic origin between patient groups, which comprised European (43%), Asian (24%),, African (17%) and South-American (16%) patients. Records of all patients withh active TB were reviewed and clinical data such as fever (rectal temperature abovee 38 °C) and anorexia (gross loss of appetite and weight loss) were scored. 14 patientss were HIV-seropositive and 67 patients were either HIV-seronegative or no antibodiess to HIV were measured. Since no differences were found between HIV-seropositive,, HIV-seronegative and patients with an unknown HIV-status (data not shown),, all patient data were combined.
ControlControl groups. Sera were obtained from 16 persons who had been in close contact
withh patients with contagious pulmonary TB; one person was tuberculin skin test positivee and 15 persons were tuberculin non-responders. Close contacts were all recrutedd from the municipal health service during contact research and did not differ fromm patient groups in age and ethnic origin. Sera were also obtained from 10 healthy controls,, matching in sex and age, all of whom were skin test negative.
Assays.Assays. Sera were collected after centrifugation and stored at -20° C until
measurements.. All assays were performed in duplicate. LBP was measured by ELISAA as described previously [8], using polyclonal rabbit anti-human LBP (5 jig/ml)) as capturing antibody, biotinylated polyclonal rabbit anti-human LBP as labelingg antibody and recombinant LBP as standard. BPI was measured with an ELISAA as described [8], using monoclonal anti-human antibody (3 \ig/m\) as capturingg antibody, biotinylated polyclonal rabbit anti-human BPI IgG as detecting antibodyy and recombinant human BPI as standard. sCD14 was measured by an ELISAA according to the instructions of the manufacturer (Biosource Europe, Fleurus, Belgium).. Detection limits of assays were, 781 pg/ml (LBP), 391 pg/ml (BPI) and 2 ng/mll (sCD14).
StatisticalStatistical analysis. All values are presented as medians (range). Comparisons
betweenn groups were made using the Wilcoxon rank-sum test for unmatched samples.. Two-sided P-values below .05 were considered significant.
Results s
LBP.LBP. Serum LBP concentrations did not differ between patients with pulmonary and
extrapulmonaryy TB (85.1(12.3-411.0) and 71.5(25.4-334.0) ng/ml respectively, NS). Patientss with active TB had higher levels (78.6(12.3-3340.0) u,g/ml) than patients duringg therapy (32.7(8.8-187.0) ng/ml, P = .001), patients who had completed therapyy (29.0(10.6-268.0) ug/ml, P < .005), close contacts (40.4(12.2-150.0) u.g/ml, PP < .05) and controls (12.6(6.4-60.4) Hg/ml, P <.001) (Figure). In patients with active TBB who had fever or anorexia, LBP was significantly raised compared to patients withh a normal temperature or without anorexia respectively (Table).
BPI.BPI. Serum BPI concentrations did not differ between patients with pulmonary and
extrapulmonaryy TB (5.9(0.4-51.5) and 8.3(0.4-123.0) ug/ml respectively, NS). Patientss with active TB had higher BPI levels (6.7 (<0.4-123.0) ng/ml) when comparedd to close contacts (3.9(<0.4-0.5) ng/ml, P = .05) and to controls (1.8(0.5-8.0),, P < .005). The median serum levels of BPI in patients who had completed therapyy (4.8(<0.4-32.9) ng/ml) and in close contacts were raised when compared to controlss (P < .05 and P = .05 resp.). There was no difference in BPI between patients withh and without clinical symptoms (Table).
sCD14.sCD14. Serum sCD14 was significantly higher in extrapulmonary TB than in
pulmonaryy TB (7.2(3.2-16.2) and 5.8(2.1-13.6) ng/ml respectively, P < .05). All patientt groups had significantly higher levels of sCD14 when compared to close contacts,, but levels of sCD14 of patients during therapy did not differ from controls. Mediann serum sCD14 concentration in patients with active TB (pulmonary or extrapulmonary)) was 6.0 (2.1-16.2) Ug/ml, which was significantly higher than in patientss during therapy (4.8(<2.0-9.0), P = .01), in patients who had completed therapyy (3.4(<2.0-9.4), P < .001), in close contacts (2.6(<2.0-4.0), P_< .001) and in healthyy controls (3.5(<2.0-6.4), P < .001). Median serum level of sCD14 in patients withh active TB who had fever was significantly raised compared to patients with a normall temperature (Table).
Discussion n
Thee host immune response to TB is at least in part initiated by stimulation of inflammatoryy cells by the mycobacterial cell wall component LAM.
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Figuree 1.
Serumm concentrations of lipopolysaccharidee binding
proteinn (LBP),
bactericidal/permeability y increasingg protein (BPI) and solublee CD14 (sCD14) in patientss with active TB (n= 82), inn patients during (n=15l and afterr (n=26) treatment, in personss who had been in close contactt with contagious TB (n=16)) and in healthy controls (n=10).. Horizontal lines representt medians.
Proteinss identified as regulators of LPS activity appear critically involved in the cellularr response to LAM. We determined the serum concentrations of LBP. BPI and sCD144 in patients with various manifestations and stages of TB. All three proteins weree elevated during active TB and declined during treatment.
Increasedd serum concentrations of LBP have been reported previously in patients withh sepsis and healthy humans injected with LPS [8, 9], Our finding of elevated concentrationss of LBP in patients with active TB may have relevance for the host reactionn to TB. Indeed, LAM induces production of tumor necrosis factor and interleukinn 1(3 by the monocyte/macrophage cell line THP-1 via a CD14 dependent mechanism,, a process which is greatly enhanced by LBP [3, 4], Thus, elevated levels
Table.. Association of LBP, BPI and sCD14 with clinical symptoms during active tuberculosis s Feverr Anorexia Yess No Yes No LBPP (ug/ml) 92.3(18.9-3340.0)* 55.9(13.0-411.0) 91.2(18.5-3340.0)* 64.7(12.3-670.0) BPII (ng/ml) 6.6(0.4-123.0) 6.8(0.4-123.0) 5.5(0.4-51.5) 6.94(0.4-123.0) sCD144 (ug/ml) 7.8(2.1-16.2)* 5.4(2.7-8.5) 6.5(3.1-16.2) 5.8(2.1-15.3)
Dataa are median (range) of 82 patients with active TB. Fever was defined as rectal temperature >38°C;; anorexia as gross loss of appetite and weight loss. * = P<.05 for difference between absence andd presence of fever or anorexia.
off LBP may facilitate inflammatory reactions in patients with TB. In accordance, LBPP levels were higher in patients with fever and/or anorexia. Since LBP has also beenn found in bronchoalveolar lavage fluids of healthy humans and patients with lungg injury [10] it is likely that LBP also influences LAM bioavailability in lungs duringg pulmonary TB.
BPII is a neutrophil degranulation product that exerts bactericidal effects on gram-negativee bacteria and neutralizes LPS activity in vitro and in vivo [2]. As in patients withh sepsis and in volunteers to whom endotoxin had been administered [8, 11], serumm BPI concentrations were higher in patients with active TB, albeit no differencess were found between patients with and without fever or anorexia. Further studiess are needed to determine whether BPI can influence LAM bioactivity in a similarr way as LPS, although this seems likely considering the over 40% amino acid sequencee homology between LBP and BPI, and the shared properties of LPS and LAMM with respect to LBP/CD14 interactions [2-6].
Previouss studies have documented elevated serum concentrations of sCD14 in patientss with sepsis [8]. Also, high levels of sCD14 have been found in bronchoalveolarr lavage fluid in patients with lung disorders, including TB [12, 13]. Ourr study expands these findings to elevated serum concentrations of sCD14 in patientss with active TB. sCD14 enables responses to LPS by cells with little or no membranee bound CD 14 [2]. Of interest, patients with extrapulmonary TB had higher levelss of sCD14 compared to patients with pulmonary TB. This may indicate a decreasedd sensivity of cell types in extrapulmonary sites to LAM when compared to alveolarr cells, but further studies on regulation of LAM bioactivity by sCD14 during TBB are needed.
Wee here report that LBP, BPI and sCD14 are elevated in serum of patients with activee TB and decrease during treatment. Wether these proteins have an essential role inn mounting an inflammatory response during TB remains to be determined.
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Acknowledgements s
Wee thank Mieke Sala for technical assistance. We are indebted to Drs. J. Bruins and K.. Vos for the provision of samples from army recruits.
References s
1.. Raviglione MC, Snider DE, Jr., Kochi A. Global epidemiology of tuberculosis. Morbidity and mortalityy of a worldwide epidemic. JAMA 1995;273:220-6
2.. Ulevitch RJ, Tobias PS. Recognition of endotoxin by cells leading to transmembrane signaling. Currentt Opin Immunol 1994;6:125-30
3.. Savedra R, Jr., Delude RL, Ingalls RR, Fenton MJ, Golenbock DT. Mycobacterial lipoarabinomannann recognition requires a receptor that shares components of the endotoxin signalingg system. J Immunol 1996;157:2549-54
4.. Zhang Y, Doerfler M, Lee TC, Guillemin B, Rom WN. Mechanisms of stimulation of interleukin-11 beta and tumor necrosis factor-alpha by Mycobacterium tuberculosis components. J Clinn Invest 1993;91:2076-83
5.. Pugin J, Heumann ID, Tomasz A, Kravchenko VV. et all. CD 14 is a pattern recognition receptor. Immunityy 1994;1:509-16
6.. Hunter SW, Brennan PJ. Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannann and lipomannan of M. tuberculosis. J Biol Chem 1990;265:9272-9
7.. Juffermans N, Verbon A, van Deventer SJH, van Deutekom H, Speelman P, van der Poll T. Tumorr Necrosis Factor and Interleukin-1 inhibitors as markers of disease activity of tuberculosis. AmJRespp Crit Care Med 1998;157:1328-31.
8.. Froon AH, Dentener MA, Greve JW, Ramsay G, Buurman WA. Lipopolysaccharide toxicity-regulatingg proteins in bacteremia. J Inf Dis 1995;171:1250-7
9.. van der Poll T, Coyle SM, Levi M, et all. Effect of a recombinant dimeric tumor necrosis factor receptorr on inflammatory responses to intravenous endotoxin in normal humans. Blood 1997;89:3727-34 4
10.. Martin TR, Mathison JC, Tobias PS, et all. Lipopolysaccharide binding protein enhances the responsivenesss of alveolar macrophages to bacterial lipopolysaccharide. Implications for cytokine productionn in normal and injured lungs. J Clin Invest 1992;90:2209-19
11.. von der Mohlen MA, van der Poll T, Jansen J, Levi M, van Deventer SJ. Release of bactericidal/permeability-increasingg protein in experimental endotoxemia and clinical sepsis. Rolee of tumor necrosis factor. J Immunol 1996;156:4969-73
12.. Hoheisel G, Zheng L, Teschler H, Striz I, Costabel U. Increased soluble CD 14 levels in BAL fluidd in pulmonary tuberculosis. Chest 1995;108:1614-6
13.. Martin TR, Rubenfeld GD, Ruzinski JT, et all. Relationship between soluble CD14. lipopolysaccharidee binding protein, and the alveolar inflammatory response in patients with acute respiratoryy distress syndrome. Am J Resp Crit Care Med 1997;155:937-44
14.. Haziot A, Ferrero E, Kontgen F, et all. Resistance to endotoxin shock and reduced dissemination off gram-negative bacteria in CD14-deficient mice. Immunity 1996;4:407-14
15.. Robert S. Jack XF, Martin Bernheiden. et all. Lipopolysaccharide-binding protein is required to combatt a murine Gram-negative bacterial infection. Nature 1997;389:742-745
