Tumour - downloads.hindawi.comdownloads.hindawi.com/journals/mi/1994/971068.pdf · A. P. Massetti,...

Research Paper

Mediators of Inflammation 3, 57-60 (1994)

THE local production of tumour necrosis factor-0t (TNF0t)was evaluated in the cerebrospinal fluid (CSF) from ten

patients with tuberculous meningitis (TBM). The degreeof intrathecal immune activation was also studied byassessing the CSF levels of 2-microglobulin (2-M) andadenosine deaminase activity (ADA). Results indicate thatelevated CSF concentrations of TNF0t, 12-M and ADAwere found in all TBM patients. Moreover, TNF0t isproduced and selectively concentrated for a long period oftime, while 2-M and ADA values progressively declineduring the course of TBM. Our findings suggest that inTBM patients, after an early activation of immune cells,there is an enhanced and continuous production of TNF0tat the site of infection.

Key words: Adenosine deaminase, fl2-microglobulin, Cere-brospinal fluid, Immune activation, Tuberculous meningitis,Tumour necrosis factor-cz

Tumour necrosis factor-=production and immune cellactivation in tuberculousmeningitis

C. M. Mastroianni,cA F. Paoletti, C. Valenti,A. P. Massetti, V. Vullo and S. Delia

Institute of Infectious Diseases, University ’LaSapienza’, Policlinico Umberto I, Viale ReginaElena 331, 00161 Rome, Italy

CA Corresponding Author

Introduction

Tumour necrosis factor-o (TNFo0 is a cytokinemainly released by cells of the monocyte-macrophage lineage in response to lipopolysacchar-ide (LPS) and other immune and inflammatorystimuli. It has been recognized as a primarymediator in the pathogenesis of infection, injuryand inflammation and in the beneficial processes ofhost response. In particular, TNFe is an importantmacrophage-activating factor for antibacterial re-sistance against infections caused by facultativeintracellular organisms, such as mycobacteria.2

In experimental models it has been reported thatTNF enhances macrophage phagocitic capacityand mycobacterial killing by human macrophages.In addition, recent lines of evidence indicated thatTNF is synthesized in large amounts in pulmonarytuberculosis and it is locally concentrated at the siteof disease activity.4,s To date, little is known aboutthe intrathecal production of TNF during thecourse of tuberculous meningitis (TBM).6 There-fore, it appears to be of interest to evaluate the localproduction and release of TNFo in the cere-brospinal fluid (CSF) of patients with TBM.

In the present study, TNFo concentrations weremeasured in CSF specimens obtained from hospitalpatients on admission and during the course ofTBM. In order to determine the extent to whichcell-mediated immunity is involved in the disease,the CSF levels of two markers of immuneactivation, such as fl2-microglobulin (/2-M) andadenosine deaminase activity (ADA), were alsomeasured.

Materials and Methods

Patients: Ten patients with TBM (five males, fivefemales) admitted to the Institute of InfectiousDiseases of the University of Rome ’La Sapienza’,were enrolled in this study. The mean age was 34.4years (range, 12-55 years). TBM was diagnosed onthe basis of findings in the CSF. In six cases CSFsmears were positive by acid-fast staining and fluidcultures grew Mycobacterium tuberculosis; in theremaining patients, diagnosis was made bycompatible cytological and biochemical findings,supported by clinical features and symptomaticimprovement with antituberculous therapy. Allpatients were sero-negative for human immuno-deficiency virus (HIV) and did not have otherdisturbances of immunity.A total of 66 CSF samples was collected from

TBM patients before starting treatment andsubsequently at various intervals of time during thecourse of infection.CSF specimens were also obtained from 15

patients with non-infectious neurological disorders,including cerebrovascular disease, hydrocephalus,encephalomyelitis and epilepsy.CSF specimens, upon collection, were placed in

a refrigerated centrifuge (4C) and spun at

3 000 rpm for 10 min. Cell supernatants were storedat -80C until use.

Assayfor TNF: TNF concentrations in CSF weremeasured by a quantitative immunoenzymaticsandwich assay (Quantikine, R&D Systems, Min-neapolis, MN, USA). Samples and standards were

(C) 1994 Rapid Communications of Oxford Ltd Mediators of Inflammation. Vol 3. 1994 57

C. M. Mastroianni et al.

incubated in anti-TNF monoclonal antibody-coated polystyrene microtiter wells. A horseradishperoxidase-conjugated anti-TNF0 polyclonal anti-body was then added and allowed to bind the TNFcaptured by the first antibody, completing thesandwich. After washing, substrate solution wasadded to the wells. The reaction was stopped byaddition of 2N HiSO4 and optical density (OD) at492 nm was measured using an ELISA microreader.To estimate the amount of TNFz in the CSFsamples we constructed a standard curve by usingconcentrations of TNF ranging from 0 to

1000pg/ml. Unknown values of TNF in thesamples were determined by referring to thestandard curve and expressed as pg/ml. Thedetection limit of the assay was 4.8 pg/ml.

Assay for fie-M: /2-M concentrations were assessedby the IMx system (Abbott Laboratories, NorthChicago, IL, USA), according to the manufacturer’sinstructions. The IMx system is based on a rapidmicroparticle enzyme immunoassay (MEIA), inwhich submicron particles are coated with a mousemonoclonal antibody specific for/2-M. The use ofmicroparticles increases the assay kinetics anddecreases the assay incubation. The sensitivity ofthe IMx /2-M was calculated to be better than5 tg/1. Normal/2-M values were considered equalor less than 3 mg/1.

ADA activity measurement: ADA activity was mea-sured in fresh CSF samples after centrifugation at300 g for 10 min. ADA assay was performed at37C by using the colorimetric method of Giusti,v

based on the determination of the amount of NH3released in the reaction mixture. The enzymaticactivity was calculated by referring to a standardcurve of ammonium sulphate in buffer and was

expressed as IU/37C/1. Normal ADA values wereconsidered equal or less than 4 IU/1.Statistical analysis: The Mann-Whitney U-test wasused for statistical analysis.

Results

CSF levels of TNF, /2-M and ADA weremeasured in ten patients with TBM and in 15control subjects with non-infectious CNS disorders.Data are shown in Figure 1 and are expressed asmean + SEM. TNF concentrations in TBMpatients (155_21.62pg/ml) were significantlyhigher than those found in controls (5.69-+-0.77pg/ml) (p < 0.0001). Similarly, TBM patients hadelevated CSF values of/32-M (8.69

___1.07 mg/1) and

ADA (10.83 _+ 1.61 IU/37C/1)in comparison withthose observed in the non-TBM cases (1.34 -t- 0.12mg/1 and 1.02 0.16 IU/37C/1, respectively) (p <0.0001 for both).The kinetics of TNF0 and both j2-M and ADA

values during the course of TBM were illustratedrespectively in Figures 2 and 3.

In all patients CSF levels of TNF were low onadmission, then progressively increased and re-mained detectable for a long period of time.Antibiotic treatment did not appear to influence theTNF levels. Similarly, there was no correlationbetween the cytokine concentrations and the clinicalcourse of the disease.On the contrary,/2-M and ADA values resulted

to be elevated on admission. Later, a progressivedecline was observed. Only two patients withculture-proven TBM showed a further increase inCSF/2-M and ADA values, despite the administra-tion of antituberculous treatment. The peak of these

12-/

I0-

8-

6-

4-

2-

0-

150

O-

FIG. 1. Levels of/t2-M, ADA and TNF in the CSF samples (n 66) from TBM patients compared with those (n 15) obtained incontrols with non-infectious neurological diseases. Data are expressed as the mean

___SEM. Comparisons between groups are made by

non-parametric test (Mann-Whitney U-test) and differences in CSF levels of/Y2-M, ADA and TNF were statistically significant (p < 0.0001for all).

58 Mediators of Inflammation. Vol 3.1994

Tumour necrosis factor- in tuberculous meningitis

300

200

100

00 10 20 30 40 50

WeeksFIG. 2. Time course of TNF levels in sequential CSF samples from the ten TBM patients.

10

20 40Weeks

.0

0 10 20 30 40

WeeksFIG. 3. Time course of J2-M and ADA levels in sequential CSF samplesfrom the ten TBM patients.

immune activation markers was associated with thedevelopment of complications (hydrocephalus,hemiplegia) and an unfavourable clinical course ofmeningitis.

Discussion

Cetl-mediated immunity plays an important rolein the control of infections by M. tuberculosis. It iswidely assumed that T-lymphocytes, throughlymphokine-mediated macrophage activation, arethe major immune cells involved both in thepathogenesis and protective mechanisms in humantuberculosis.1

5’0 In the case of TBM, it is conceivable that theinvolvement of cellular immune system mainlyoccurs in the central nervous system (CNS). In thisrespect, the dosage in the CSF of two markers ofimmune activation, such as fl2-M and ADA,represents a useful tool to investigate the degree towhich cell-mediated immunity is stimulated in theCNS. /2-M is a portion of the major histocompat-ibility complex class I antigen (MHC-1) and isexpressed on the surface of lymphocytes andmacrophages. With regard to the adenosinedeaminase, the activity of this enzyme is increasedin lymphoid cells, especially during T-cell prolifera-tion and differentiation.

Previous investigations have already demon-$0 strated a close correlation between elevated CSF

ADA values and TBM,1’12 while an increase in theCSF concentrations of/2-M has been reported onlyin viral and pyogenic meningitis. 13 In the present

Mediators of Inflammation. Vol 3.1994 59

C. M. Mastroianni et al.

study, both /2-M and ADA levels were found tobe elevated in the CSF of all TBM patients, thusindicating a marked stimulation of immune systemwithin the CNS. The increased CSF levels of/2-Mwere possibly a consequence of cell-mediatedcytotoxicity directed against macrophages infectedby M. tuberculosis. 14 The elevation of CSF ADAvalues was most likely related to the local immuneresponse as the result of proliferating lymphocytesin response to mycobacterial antigen.

Intrathecal immune activation appears to be alsoassociated with increased cytokine expression in theCNS. Indeed, our results showed that TBM patientshad significantly higher CSF TNFo concentrationsthan those found in control subjects withnon-infectious neurological diseases. The localproduction of TNF is low in the initial phase ofTBM, while the peak in the CSF concentration wasobtained later. Moreover, the CSF levels of thiscytokine did not decrease during the course of thedisease, but they were elevated for a long time inall patients, irrespective of the antibiotic therapyand the clinical course of TBM. This pattern is quitedifferent from that observed in the case of/2-M andADA, whose levels declined rapidly during thecourse of the disease. Only two patients whodeveloped neurological complications showed afurther increase in the CSF/2-M and ADA values.

Taken together, our findings suggest that a greatstimulation of immune T-cells primarily occurs inthe acute phase of TBM and may account for theearly increase in the CSF /2-M and ADA values.On the contrary, the enhancement of cytokineexpression related to TNF production is a laterbut more prolonged phenomenon. It is likely thatbrain macrophages activated by T-cell-mediatedpathway are the source of TNFo in TBM patients.This hypothesis is also supported by previousinvestigations that have demonstrated that M.tuberculosis cell wall components can trigger therelease of TNFo from human and murinemacrophages, as well as from pleural fluidmononuclear cells of patients with pulmonarytuberculosis. 5,15,16

In conclusion, our data provide direct evidencethat TNFo is produced and selectively concentrated

for a long period of time in the CSF from patientswith TBM. The chronic release of TNFo at the siteof the infection suggests that this cytokine may beinvolved in the complex immunoregulatory mech-anism that contributes to mycobacterial contain-ment and elimination.

References

1. Rosenblum MG, Donato NJ. Tumor necrosis factor : multifacetedpeptide hormone. Crit Rev Immunol 1989; 9: 21-44.

2. Havell EA. Evidence that tumor necrosis factor has important role inantibacterial resistance. J Immunol 1989; 143: 2894-2899.

3. Bermuduz LEM, Young LS. Tumor necrosis factor, alone in combinationwith IL-2, but not IFN-gamma, is associated with macrophage killing ofMycobacterium avium complex. J Immunol 1988; 140: 3006.

4. Barnes PF, Fong S-E, Brennan Pj, Twoney PE, Mazumder A, Modlin RL.Local production of tumor necrosis factor and IFN-y in tuberculous pleuritis.J Immuno11990; 145: 149-154.

5. Takashima T, Ueta C, Tsuyuguchi I, Kishimoto S. Production of tumor

necrosis factor alpha by monocytes from patients with tuberculous pleuritis.Infect Immun 1990; 58: 3286-3292.

6. Glimaker M, Kragsbjerg P, Forsgren M, Olc&n P. Tumor necrosis factor-(TNF00 in cerebrospinal fluid from patients with meningitis at differentetiologies: high levels of TNF indicate bacterial meningitis. J Infect Dis1993; 167: 882-889.

7. Giusti G. Adenosine deaminase. In: Bergmeyer HU, ed. Methods in EnwmaticAnalysis. Wienhen: Verlag Chemie, 1974; 1092-1099.

8. Leveton C, Barnass B, Champion B, et al. T-cell mediated protection in mice

against virulent Mycobacterium tuberculosis. Infect Immun 1989; 57: 390-395.9. Ellner J, Wallis RS. Immunologic aspects of mycobacterial infections. Rev

Infect Dis 1989; 2 (Suppl 2): S455-S459.10. Orme IM, Miller ES, Robert AD, et al. T-lymphocytes mediating protection

of mice against cytolysis during the course of Mycobacterium tuberculosisinfection. Evidence for different kinetics and recognition of wide spectrumof protein antigens. J Immuno11992; 148: 189-196.

11. Malan C, Donald PR, Golden M, Taljaard jjF. Adenosine deaminase levelsin cerebrospinal fluid in the diagnosis of tuberculous meningitis. J Trop MedHyg 1984; 87: 33-40.

12. Ribera E, Martinez-Vazquez JM, Ocana I, Segura R, Pascual C. Activity ofadenosine deaminase in cerebrospinal fluid for the diagnosis and follow-upof tuberculous meningitis in adults. J Infect Dis 1987; 155: 603-607.

13. Adachi N. Beta-2-microglobulin levels in the cerebrospinal fluid: their valuedisease marker. Eur Neurol 1991; 31: 181-185.

14. Wallis RS, Vjecha M, Amir-Tahmasseb M, et al. Influence of tuberculosishuman immunodeficiency virus (HIV-1): enhanced cytokine expression

and elevated fle-microglobulin in HIV-1 associated tuberculosis. J Infect Dis1993; 167: 43-48.

15. Moreno C, Taverne J, Mehlert A, et al. Lipoarabinomannan fromMycobacterium tuberculosis induces the production of tumour necrosis factorfrom human and murine macrophages. Clin Exp Immuno11989; 76: 240-245.

16. Barnes PF, Chatterjee D, Abrams JS, et al. Cytokine production induced byMycobacterium tuberculosis lipoarabinomannan. Relationship to chemicalstructure. J Immuno11992; 149: 541-547.

ACKNOWLEDGEMENTS. This work supported in part by grant fromM.U.R.S.T. (40% 1991), Rome, Italy. We thank Dr Pietro Gallo (ISS COA,Rome) for help in statistical analysis.

Received 5 November 1993"accepted in revised form 7 December 1993

60 Mediators of Inflammation. Vol 3.1994

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