of March 21, 2018.This information is current as

of Peripheral or Local Immune ResponsesNot Associated with Measurable AttenuationDexamethasone in Tuberculous Meningitis Is The Clinical Benefit of Adjunctive

Hien and Jeremy FarrarThi Dung, Kasia Stepniewska, Nicholas J. White, Tran TinhQuyen, Tran Thi Hong Chau, Pham Phuong Mai, Nguyen Cameron P. Simmons, Guy E. Thwaites, Nguyen Than Ha

http://www.jimmunol.org/content/175/1/579doi: 10.4049/jimmunol.175.1.579

2005; 175:579-590; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/175/1/579.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Clinical Benefit of Adjunctive Dexamethasone inTuberculous Meningitis Is Not Associated with MeasurableAttenuation of Peripheral or Local Immune Responses1

Cameron P. Simmons,2,3* Guy E. Thwaites,3* Nguyen Than Ha Quyen,3*Tran Thi Hong Chau,3† Pham Phuong Mai,3† Nguyen Thi Dung,3† Kasia Stepniewska,3‡

Nicholas J. White,3‡ Tran Tinh Hien,3† and Jeremy Farrar3*

Outcome from tuberculous meningitis (TBM) is believed to be dependent on the severity of the intracerebral inflammatoryresponse. We have recently shown that dexamethasone improved survival in adults with TBM and postulated that the clinicaleffect would be associated with a measurable systemic and intracerebral impact on immunological markers of inflammation.Prolonged inflammatory responses were detected in all TBM patients irrespective of treatment assignment (placebo or dexameth-asone). The inflammatory response in the cerebrospinal fluid was characterized by a leukocytosis (predominantly CD3�CD4� Tlymphocytes, phenotypically distinct from those in the peripheral blood), elevated concentrations of inflammatory and anti-inflammatory cytokines, chemokines, and evidence of prolonged blood-brain barrier dysfunction. Dexamethasone significantlymodulated acute cerebrospinal fluid protein concentrations and marginally reduced IFN-� concentrations; other immunologicaland routine biochemical indices of inflammation were unaffected. Peripheral blood monocyte and T cell responses to Mycobac-terium tuberculosis Ags were also unaffected. Dexamethasone does not appear to improve survival from TBM by attenuatingimmunological mediators of inflammation in the subarachnoid space or by suppressing peripheral T cell responses to mycobac-terial Ags. These findings challenge previously held theories of corticosteroid action in this disease. An understanding of howdexamethasone acts in TBM may suggest novel and more effective treatment strategies. The Journal of Immunology, 2005, 175:579–590.

C linical outcome from tuberculous meningitis (TBM),4 themost lethal form of infection with Mycobacterium tuber-culosis, is believed to be dependent on the severity of the

host intracerebral inflammatory response (1). Corticosteroids areused in a wide variety of inflammatory conditions and provideclinical benefit by apparently diverse and incompletely understoodmechanisms. They have long been considered for the adjunctivetreatment of TBM, although evidence of their effect on morbidityand mortality has been difficult to obtain (2). Previous studies sug-gested corticosteroids attenuated the inflammatory response in thesubarachnoid space of those with TBM (cerebrospinal fluid con-centrations of leukocytes, protein, and glucose returned to normalfaster in corticosteroid-treated patients) but were too small to dem-onstrate a clear effect on survival, and measurement of molecular

inflammatory mediators was impossible (3). Recent controlled tri-als provided stronger evidence that adjunctive corticosteroids im-prove survival in children with TBM (4). Cerebrospinal fluid con-centrations of protein, globulin, and glucose normalized faster inthe corticosteroid-treated children without a significant effect oncerebrospinal leukocyte counts; other inflammatory mediators con-sidered important in the pathogenesis of TBM, such as TNF-� (1),were not measured (5).

We recently performed a controlled trial of adjunctive dexa-methasone in 545 Vietnamese adults with TBM that demonstrateddexamethasone improved survival but did not prevent severe dis-ability (6). We postulated that the effect of dexamethasone on sur-vival would be associated with a measurable anti-inflammatoryresponse in the subarachnoid space with evidence of systemic at-tenuation of response to M. tuberculosis Ags. Therefore, we com-pared the kinetics of the inflammatory response in cerebrospinalfluid and peripheral blood from 87 adults with TBM randomlyassigned to treatment with adjunctive dexamethasone or placebo.Our aim was to define a mechanism of action for the effect ofdexamethasone on survival and to identify molecules and cellscritical to the pathogenesis of TBM that might suggest novel andmore specific treatments.

Materials and MethodsStudy participants and setting

A randomized, double blind, placebo-controlled trial of adjunctive dexa-methasone for the treatment of TBM was performed in 545 adults betweenApril 2001 and April 2003 according to methods described previously (6).Study participants were recruited from two centers: Pham Ngoc ThachHospital for tuberculosis and the Hospital for Tropical Diseases (HTD) inHo Chi Minh City, Vietnam. The immunomodulatory effects of dexameth-asone were only studied in adults treated at HTD (n � 93). Adults (�14

*Oxford University Clinical Research Unit, Hospital for Tropical Diseases, and †Hos-pital for Tropical Diseases, Ho Chi Minh City, Vietnam; and ‡Faculty of TropicalMedicine, Mahidol University, Bangkok, Thailand

Received for publication December 15, 2004. Accepted for publication April14, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Wellcome Trust, United Kingdom.2 Address correspondence and reprint requests to Dr. Cameron P. Simmons, OxfordUniversity Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu,District 5, Ho Chi Minh City, Vietnam. E-mail address: csimmons@hcm.vnn.vn3 All authors helped design the study and collect the data. C.P.S., N.T.H.Q., and K.S.analyzed the data. C.P.S., G.E.T., and J.F. wrote the paper, with review and commentsfrom all authors.4 Abbreviations used in this paper: TBM, tuberculous meningitis; PPD, purified pro-tein derivative; SFU, spot-forming unit; CFP, culture filtrate protein; WCL, whole-cell lysate; GC, glucocorticoid; ESAT-6, early secreted Ag target.

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years of age) with definite, probable, or possible TBM were eligible toenter the trial. Definite TBM was defined as clinical meningitis (nuchalrigidity and abnormal cerebrospinal fluid parameters) and acid-fast bacilliseen, or M. tuberculosis cultured, from the cerebrospinal fluid. ProbableTBM was defined as clinical meningitis and one or more of the following:suspected active pulmonary tuberculosis on chest radiography; acid-fastbacilli found in any other specimen; clinical evidence of other extrapul-monary tuberculosis. Possible TBM was defined as clinical meningitis andat least four of the following: history of previous tuberculosis; predomi-nance of lymphocytes in the cerebrospinal fluid; illness duration �5 days;cerebrospinal fluid-blood glucose �0.5; altered consciousness; yellow ce-rebrospinal fluid; and focal neurological signs. All adults were tested forAbs to HIV 1/2.

The ethical and scientific committees of both hospitals, the Health Ser-vices of Ho Chi Minh City, and the Oxford Clinical Research Ethics Com-mittee approved the study protocol. Written informed consent to participatein the study was obtained from all patients or their relatives.

Treatment and specimen collection

Adults were allocated randomly to start dexamethasone sodium phosphateor placebo (VIDIPHA) as soon as possible after the start of four antitu-berculosis drugs. Adults with grade II or III disease received an i.v. drugfor 4 wk (wk 1, 0.4 mg/kg/day; wk 2, 0.3 mg/kg/day; wk 3, 0.2 mg/kg/day;wk 4, 0.1 mg/kg/day), then 4 wk of an oral drug starting at 4 mg total perday, reducing each week by 1 mg until zero. Adults with grade I diseasereceived 2 wk of an i.v. drug (wk 1, 0.3 mg/kg/day; wk 2, 0.2 mg/kg/day),then 4 wk of an oral drug (wk 3, 0.1 mg/kg/day orally; then 3 mg total perday reducing by 1 mg each week until zero).

Serial, paired cerebrospinal fluid and peripheral blood samples werecollected before and after starting the study drug as part of normal clinicalcare. When possible, paired samples were collected on days 3, 7, 30, 60,and 270 after the commencement of treatment. In practice, there was vari-ation in the time of sampling; details are provided for the period patientsreceived the study drug. Over a period of 9 mo, we collected a total of 228cerebrospinal fluid samples from 41 patients in the placebo arm and 279cerebrospinal fluid samples from 46 patients in the dexamethasone arm.There were pretreatment samples from 41 patients in the placebo arm and45 patients in the dexamethasone arm. In the first 4 days of treatment, therewere samples from 22 patients in the placebo arm (median time, 3 days)and samples from 30 patients in the dexamethasone arm (median time, 3days). Between days 5 and 12, there were samples from 33 patients in theplacebo arm (median time, 7 days) and samples from 39 patients in thedexamethasone arm (median time, 7 days). Between days 13 and 20, therewere samples from 8 patients in the placebo arm (median time, 14 days)and samples from 12 patients in the dexamethasone arm (median time, 28days). Between days 21 and 35, there were samples from 34 patients in theplacebo arm (median time, 28 days) and samples from 44 patients in thedexamethasone arm (median time, 28 days).

Routine clinical investigations, flow-cytometric analysis of cellular phe-notype, and functional studies were performed on the same day the spec-imen was taken; all other measurements were performed on aliquots ofcerebrospinal fluid supernatant frozen at �70°C. Cerebrospinal fluid su-pernatants samples were typically frozen within 3 h of collection.

Assessment of the clinical and inflammatory response totreatment

The primary clinical outcome was death or severe disability 9 mo after ran-domization. Local and peripheral immune responses were assessed blind toclinical outcome and treatment allocation by the following methods.Routine cerebrospinal fluid measurements. Concentrations of total anddifferential cerebrospinal fluid leukocytes, lactate, glucose, and proteinwere measured by standard methods.Cytokine and chemokine measurements and assessment of the blood-brain barrier. Cytokines (IL-6, IL-10, IL-1�, TNF, IL-8, and IL-12p70)and chemokines (IP-10, MCP-1, RANTES, and Mig) were measured usinga cytometric bead array assay (BD Biosciences) according to the manu-facturer’s instructions, except all samples were fixed in 4% paraformalde-hyde before analysis. The mean limits of detection for the cytokines andchemokines were as follows: IL-6, 4.3 pg/ml; IL-10, 4.8 pg/ml; IL-1�, 2.8pg/ml; TNF, 4.2 pg/ml; IL-8, 5 pg/ml; IL-12p70, 5.1 pg/ml; IP-10, 6 pg/ml;MCP-1, 5.4 pg/ml; RANTES, 4.6 pg/ml; MIG, 4.8 pg/ml. All measure-ments were performed in duplicate, and the results are expressed as themean value. To control for interassay variation, three different control sam-ples (derived from pooled cerebrospinal fluid) were tested in each andevery assay to gauge the extent of interassay variation. The maximuminterassay variation recorded for any one cytokine was 12.3%. The max-

imum interassay variation recorded for any one chemokine was 32.7%.Blood-brain barrier integrity was assessed by measurement of paired ce-rebrospinal fluid and plasma albumin concentrations by standard methodswith calculation of the albumin index using the formula [albumincsf]/[al-buminplasma] (7).Flow-cytometric analysis of cerebrospinal fluid and peripheral blood leu-kocyte phenotypes. Flow-cytometric analysis of whole blood and cerebro-spinal fluid stained with fluorochrome-conjugated mAbs (BD Biosciences)was performed on a FACSCalibur flow cytometer (BD Biosciences). Theantagonist anti-CD95 mAb ZB4 (Beckman Coulter) was used at a finalconcentration of 5 �g/ml. CD3� T cells were purified from PBMCs andcerebrospinal fluid using Ab-coated magnetic beads according to the man-ufacturer’s instructions (Miltenyi Biotec). T cells were activated by cocul-ture with PMA (Sigma-Aldrich) and ionomycin (Sigma-Aldrich) and usedat final concentrations of 10 ng/ml and 1 �g/ml, respectively.Peripheral blood responses to M. tuberculosis Ags. Ex vivo responseswere assessed to defined Ags of M. tuberculosis on serial peripheral bloodsamples using a whole-blood assay and an IFN-� ELISPOT. The whole-blood assay incubated heparinized, fresh whole blood in duplicate 0.5-mlvolumes with recombinant early secreted Ag target-6 (rESAT-6) (5 �g/ml),H37Rv culture filtrate proteins (CFPs) (5 �g/ml), H37Rv whole-cell lysate(WCL) (5 �g/ml) (all obtained from the University of Colorado), PBS(negative control), and the positive control, PHA (5 �g/ml) (Sigma-Al-drich). Supernatants were removed after a 48-h incubation at 37°C with 5%CO2 in air and frozen at �20°C until analyzed for their cytokine content.Measurement of IFN-� was performed by capture ELISA using a com-mercial assay kit (BD Biosciences) and performed according to the man-ufacturer’s instructions. Monocyte-derived cytokines (IL-6, IL-10, IL-1�,TNF, IL-8, and IL-12p70) were measured using a cytometric bead arrayassay (BD Biosciences) according to the manufacturer’s instructions. Cy-tokine concentrations detected in whole-blood cultures stimulated withPBS were subtracted from values detected in Ag or mitogen-stimulatedcultures.

IFN-� ELISPOT assays were used to measure the frequency of T cellsproducing IFN-� in response to rESAT-6, purified protein derivative(PPD), and 15 15-mer peptides spanning the M. tuberculosis ESAT-6 Ag.The assay was performed according to the manufacturer’s instructions(Mabtech). Briefly, a total of 1–3 � 105 PBMCs was added in 100 �l ofculture medium per well together with PPD (5 �g/ml), rESAT (5 �g/ml),pooled ESAT-6 peptides (final concentration, 1 �g/ml), or the positivecontrol, PHA (5 �g/ml). After overnight incubation and assay develop-ment, the number of spot-forming units (SFU) in each well was countedusing a dissecting microscope, and the background (no Ag stimulation) wassubtracted. An assay was valid only if 1) wells containing PBMCs stim-ulated with PHA were confluent with IFN-� SFU and 2) there were twiceas many SFU in Ag-stimulated wells as negative control wells. The medianbackground frequency of IFN-� SFU was 6 SFU/million PBMCs (range,0–328).

Statistics

The methods for primary and stratified subgroup analysis of death anddisability were reported previously (6). For the purposes of this study,adults were excluded from the analysis if they stopped the study drug earlyfor any reason or an alternative diagnosis was confirmed. Continuous vari-ables were compared by Student’s t test if normally distributed and byMann-Whitney U test if not normally distributed. Categorical variableswere compared by the �2 test (or Fisher’s exact test when appropriate). Allp values were two-sided. The magnitude of change from baseline of in-flammatory indices was assessed in patients who had paired cerebrospinalfluid samples collected �2 days before receiving the study drug and be-tween 1 and 7 days after beginning therapy. The magnitude of change ineach parameter was determined by subtracting the value measured in thefirst week of therapy from the value obtained before treatment. Kinetics ofserial measurements in individual patients were summarized using areaunder the curve, calculated per unit time of study drug administration, bythe trapezoid method. All analyses were performed using SPSS version 10(SPSS) and STATA 5 (StataCorp).

ResultsBaseline variables and impact of dexamethasone on clinicaloutcome

Ninety-three patients were recruited to the trial of dexamethasoneat the HTD (6). We excluded 6 of 93 patients from subsequentanalysis of immune responses because they withdrew consent (n �1), had medical contraindications that resulted in withdrawal of the

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study drug earlier than prescribed (n � 4), or had a confirmedalternative diagnosis (n � 1). The baseline clinical characteristicsof 87 patients randomized to dexamethasone (n � 46) or placebo(n � 41) included in this study are shown in Table I.

Dexamethasone treatment was associated with a reduced risk ofdeath (relative risk, 0.69; 95% confidence interval, 0.52–0.92; p �0.01) by intention-to-treat analysis of all patients (n � 545). Treat-ment effect was homogenous (test of heterogeneity, p � 0.05)across subsets defined by disease severity grade (stratified relativerisk of death, 0.68; 95% confidence interval, 0.52–0.91; p �0.007), HIV infection (stratified relative risk of death, 0.78; 95%confidence interval, 0.59–1.04; p � 0.08), and hospital of admis-sion (stratified relative risk of death, 0.70; 95% confidence inter-val, 0.53–0.92; p � 0.01) (6).

Impact of dexamethasone on routine cerebrospinal fluidmeasurements and the integrity of the blood-brain barrier

Collection of paired cerebrospinal fluid samples immediately be-fore treatment and during the first week of therapy allowed us tomeasure the magnitude of change from baseline in a range of rou-tine and immunological markers of inflammation. Dexamethasoneelicited significantly greater improvements in cerebrospinal fluidtotal protein concentration during the first week of therapy(mean � SD; net reduction with dexamethasone (n � 33) 0.64 �1 mg/ml vs increase with placebo (n � 39) 0.14 � 1.4 mg/ml; p �0.01). Dexamethasone did not significantly alter the magnitude ofchange from baseline of other cerebrospinal fluid parameters, in-cluding lymphocytes, neutrophils, lactate, opening pressure, thecerebrospinal fluid-plasma glucose ratio, or the albumin index dur-ing the first week of therapy compared with placebo (data notshown). Additional stratification and analysis of values collected inthe first week of therapy (e.g., baseline vs days 1–3 and baseline vsdays 4–7) did not reveal any additional differences between theplacebo and steroid arms.

Comparison of routine variables over the 2-mo interval of studydrug administration and the overall 9-mo treatment period sug-gested dexamethasone did not impact on the kinetics of responses(Fig. 1). Comparison of the area under the curve per unit time foreach variable during the 6- to 8-wk period of study drug admin-istration also indicated dexamethasone did not significantly mod-ulate cerebrospinal fluid responses (data not shown).

Impact of dexamethasone on cerebrospinal fluid cytokine andchemokine concentrations

Dexamethasone elicited a reduction in cerebrospinal fluid IFN-�concentrations during the first week of therapy (mean � SD; netreduction with dexamethasone (n � 27) 2.2 � 3.3 ng/ml vs netreduction with placebo (n � 25) 2.0 � 5.9 ng/ml), but this wasmarginally not significant ( p � 0.06). In the first week of therapy,dexamethasone did not significantly alter the magnitude of changefrom baseline of the other cerebrospinal fluid cytokines or chemo-kines including IL-6, IL-8, IL-10, RANTES, MCP-1, Mig, andIP-10 during the first week of therapy compared with placebo (datanot shown). Dexamethasone also did not significantly alter thekinetics of any of the cerebrospinal fluid cytokines or chemokinesover the 2-mo period of study drug administration (Fig. 2). Con-centrations of IFN-�, IL-6, IL-8, and IL-10 fell slowly, with allcytokines remaining detectable in most patients for at least 2 mo(Fig. 2). TNF was detected in a majority of pretreatment cerebro-spinal fluid samples (44 of 61 samples; range, 2.8–301 pg/ml), butits concentration fell rapidly with treatment without detectable in-fluence of dexamethasone (Fig. 2). IL-12p70 (3 of 61) and IL-1�(7 of 61) were seldom detected in pretreatment cerebrospinal fluidsamples, and dexamethasone did not significantly alter the fre-quency with which they were detected after initiation of therapy(data not shown). The cerebrospinal fluid contained significantconcentrations of the chemokines RANTES, Mig, IP-10, andMCP-1 (Fig. 3). Their concentration was maximal before treat-ment, with IP-10 and Mig, in particular, present at high concen-trations. Dexamethasone failed to significantly alter the kinetics ofany of these molecules (Fig. 3).

Impact of dexamethasone on the kinetics and phenotype ofcerebrospinal fluid and PBLs

Antituberculosis therapy was associated with a steady rise in theabsolute number of CD3�CD4� and CD3�CD8� T cells andCD16�56� NK cells in the peripheral blood of TBM patients in-dependent of treatment assignment (Fig. 4). CD3� T cells were thedominant lymphocyte subset present in cerebrospinal fluid (Fig. 4).Acutely (first 2 wk of therapy), CD3� T cells were significantlyoverrepresented ( p � 0.001) as a percentage of lymphocytes in thecerebrospinal fluid compared with the peripheral blood of the same

Table I. Baseline clinical variables of 87 adults with TBM randomized to adjunctive dexamethasone or placebo and included in the investigation ofimmune responses

Variable

Allocated Dexamethasone (n � 46) Allocated Placebo (n � 41)

n/Median %/Range n/Median %/Range

Males 24 52.2 23 56.1Age (years) 26.5 15–68 32 16–79Diagnosis on discharge

Confirmed TBM 33 71.7% 23 56.1%Probable TBM 5 10.9% 11 26.8%Possible TBM 8 17.4% 7 17.1%

BCG scara 3 10.3 1 3.8Duration of symptoms (days) 17 5–151 14 5–60Weight (kg) 47 30–75 49 30–65Temperature on admission (°C) 38.9 37–40.6 38.5 37–40.5Glasgow coma score (/15) 14 6–15 14 3–15MRC grade

1 16 34.8 11 26.82 18 39.1 19 46.33 12 26.1 11 26.8

HIV infected 1 2.2 1 2.4HIV not tested 1 2.2 0

a Missing data from 16 in the dexamethasone group and 17 in the placebo group.

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FIGURE 1. Dynamics of changes in cerebrospinal fluid leukocyte populations and cerebrospinal fluid metabolic indices in adult TBM patients ran-domized to dexamethasone or placebo. The data show changes in the means (�95% confidence interval) of the absolute count of cerebrospinal fluid whitecells (A), percentage of neutrophils (B), percentage of lymphocytes (C), total protein concentration (D), cerebrospinal fluid albumin ratio (expressed as apercentage of the serum albumin concentration) (E), ratio of the cerebrospinal fluid-blood glucose concentration (F), cerebrospinal fluid lactate concen-tration (G), and opening pressure at the lumbar puncture (H).

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patient (mean � SD; 76.2 � 12.2% in cerebrospinal fluid vs62.8 � 9.9% in paired blood (n � 18 patients)), suggesting theseT cells were selectively recruited or retained in the cerebrospinalfluid. The absolute numbers of lymphocyte subsets in cerebrospi-nal fluid did not change significantly during the first 2 mo of ther-apy (Fig. 4), because although the total numbers of leukocytes fell,the proportion of lymphocytes increased (Fig. 1C). Dexametha-

sone therapy did not alter the kinetics of absolute numbers (Fig. 4)or percentages (data not shown) of any of the cerebrospinal fluid orPBL subsets.

Given the prominence of CD3�CD4� T cells in the cerebrospi-nal fluid, we investigated their phenotype further. The majority ofCD3�CD4� T cells were CD45RA�CD45RO�CD95�CD38�

and expressed an �� TCR (Fig. 5). These cells also universally

FIGURE 2. Dynamics of changes in cerebrospinal fluid inflammatory and anti-inflammatory cytokines in adult TBM patients randomized to dexameth-asone or placebo. The data depict changes over time in mean (�95% confidence interval) cerebrospinal fluid concentrations of IL-8 (A), IL-10 (B), IFN-�(C), TNF (D), and IL-6 (E). The limit of detection for each cytokine is described in Materials and Methods.

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expressed the chemokine receptor CCR5 but not CCR7 (data notshown). Cerebrospinal fluid CD3�CD4� T cells were phenotypi-cally distinct from peripheral blood CD3�CD4� cells from thesame patient, which were rarely CD45RA�CD45RO� and ex-pressed CD95� at lower frequencies (Fig. 5). In cerebrospinal fluidcollected during the first 2 wk of therapy from 10 patients,CD3�CD4�CD45RA�CD45RO� cells represented, on average,77% (range, 51–91%) of the CD3�CD4� T cell population com-pared with 8.7% (range, 3–16%) in the paired blood sample. Thus,the majority of CD4� lymphocytes in the cerebrospinal fluid par-adoxically coexpressed surface molecules characteristic of naive(CD45RA�) and activated memory (CCR5�CCR7�CD45RO�) Tcells. The majority of cerebrospinal fluid CD3�CD8� T cells alsoexpressed an activated phenotype (CD38�CD95�) (Fig. 5C).Dexamethasone did not influence the absolute number or pheno-types of these cells.

Functional phenotype of cerebrospinal fluid T cells

Because cerebrospinal fluid T cells are in close proximity to thesite of disease, we attempted to define their functional phenotypeand determine whether they were a relevant source of IFN-� in thecerebrospinal fluid. To do this, we purified cerebrospinal fluid andperipheral blood CD3� T cells from six TBM patients in the first2 wk of therapy (three patients on placebo and three patients ondexamethasone; median, 7 days). Ex vivo culture of cerebrospinal

fluid T cells (105 per well) for 48 h with or without mitogen ac-tivation (PMA/ionomycin), failed to elicit production of detectablelevels of IFN-� in culture supernatants. In contrast, peripheralblood CD3� T cells (105 per well) secreted detectable quantities ofIFN-� (mean, 623 pg/ml; range, 230-1204) after mitogen stimu-lation. Similar results were obtained when flow cytometry orIFN-� ELISPOT were used to assess IFN-� production; only mi-togen-activated peripheral blood CD3� T cells, but not paired ce-rebrospinal fluid CD3� T cells, yielded IFN-�-producing cells(data not shown).

To determine whether mitogen incubation induced activation-induced cell death in cerebrospinal fluid T cells, the viability ofcells was measured before and after a 4-h mitogen stimulation. Inresponse to mitogen, the viability of cerebrospinal fluid CD3� Tcells dropped 4-fold vs a modest drop in the viability of peripheralblood CD3� T cells (Fig. 6).

To examine whether the cell death-signaling receptor CD95(FAS) participated in activation-induced cell death, the anti-CD95mAb ZB4, which inhibits CD95 signaling and apoptosis, was usedin activation experiments. Cerebrospinal fluid CD3� T cells wereisolated from six samples derived from five patients (three patientson placebo and two patients on dexamethasone) in the first 2 wk oftherapy. CD3� T cells were cultured for 4 h with PMA/ionomycinin the presence and absence of anti-CD95 mAb ZB4 or an isotypecontrol Ab. The mean reduction in viability of mitogen-activated

FIGURE 3. Dynamics of changes in cerebrospinal fluid chemokines in adult TBM patients randomized to dexamethasone or placebo. The data depictchanges over time in mean (�95% confidence interval) cerebrospinal fluid concentrations of IP-10 (A), Mig (B), MCP-1 (C), and RANTES (D). The limitof detection for each chemokine is described in Materials and Methods.

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FIGURE 4. Dynamics of changes in absolute counts of lymphocyte subsets in cerebrospinal fluid and peripheral blood of TBM patients randomized todexamethasone or placebo. Shown are concentrations of CD3�CD4� T cells, CD3�CD8� T cells, CD16�CD56� NK cells, and CD19� B cells. Data forcerebrospinal fluid (A, C, E, and G) are shown for samples collected within the first 2 mo of therapy. Data for peripheral blood (B, D, F, and H) are shownfor 9 mo of therapy. All data represent the mean (�95% confidence interval) of absolute counts at different time points.

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FIGURE 5. Phenotype of cerebrospinal fluid and peripheral blood T cells in TBM patients. The phenotype of cerebrospinal fluid T cells were definedin 10 TBM cases (five from each study arm). Shown is representative flow cytometry data acquired from the cerebrospinal fluid and blood of a patient withTBM grade II 10 days after beginning therapy. In A, the percentage of CD3�CD4� T cells in the cerebrospinal fluid and blood bearing either �� or ��T cell receptors or CD45RA/RO (B) is shown. In C and D, the percentage of CD3�CD8� or CD3�CD4� T cells, respectively, bearing CD38 or CD95is shown. Isotype control Abs were used to establish gating parameters.

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cerebrospinal fluid CD3� T cells cultured with anti-CD95 was notsignificantly different (67 � 16%) from T cells cocultured with anisotype control Ab (61 � 19%). These data suggest activation-induced cell death of cerebrospinal fluid T cells occurred indepen-dently of CD95.

Impact of dexamethasone on peripheral blood responses toM. tuberculosis Ags

Whole-blood cultures from patients randomized to dexamethasoneor placebo were used to measure ex vivo T cell and monocyteresponses to mitogen and Ag stimulation. The advantage of thisapproach is that it measures cellular responsiveness in the presenceof physiological levels of the study drug. Dexamethasone did notmeasurably influence the concentrations of monocyte-derived cy-tokines elicited by challenge of whole-blood cultures with a celllysate of M. tuberculosis H37Rv (Table II). This suggests the ca-pacity of monocytes to mount cytokine responses ex vivo to my-cobacterial Ags was unimpaired in the presence of dexamethasone.Whole-blood assays were also used to detect T cell responses,measured via IFN-� production, to rESAT-6, CFPs, and a WCL ofM. tuberculosis (Fig. 7). The concentration of IFN-� elicited bymitogen stimulation was suppressed early in dexamethasone-treated patients compared with placebo-treated patients (Fig. 7),

although comparison of area under the curve between the twotreatment arms was marginally not significant ( p � 0.06). Dexa-methasone did not significantly alter the T cell responses toESAT-6, CFPs, or WCL (Fig. 7), although the number of samplesexamined was small.

IFN-� ELISPOT assays

Responses in PBMCs were measured to PPD, rESAT-6, or over-lapping peptides spanning ESAT-6 (Fig. 8). ELISPOT frequenciesto all Ags were generally lowest during the acute stage of illness,particularly against PPD, but increased with time. Responses toPPD were strongest 6–10 wk after commencement of therapy butthereafter waned. Responses to rESAT-6 and to overlapping pep-tides spanning ESAT-6 varied, to a lesser extent, and remaineddetectable after 9 mo of therapy. The magnitude of the IFN-�ELISPOT responses to these Ags was independent of treatmentassignment, although the number of subjects studied was small.

DiscussionWe have recently performed a randomized, controlled trial of ad-junctive dexamethasone for the treatment of TBM in 545 Viet-namese adults that showed dexamethasone improved survival butfailed to reduce severe disability after 9 mo of treatment (6). Thisstudy involved a representative subset of adults recruited to thetrial and tested the long-standing assumption that the clinical ben-efits are consequent to the broad immunosuppressive actions ofdexamethasone, particularly those acting on the meninges and thesubarachnoid space. However, the pathogenesis of TBM is poorlyunderstood, and the mechanism for the effect of dexamethasone onsurvival, in this and other infections of the CNS, notably pyogenicbacterial meningitis, remains unknown. The small numbers, theinability to analyze serial samples, and the limited availability oftechniques capable of assessing the cellular and molecular immuneresponse have limited previous studies. This study was allied to alarge clinical trial and combined carefully recorded clinical datawith a breadth of laboratory techniques investigating the intrace-rebral and extracerebral inflammatory response to M. tuberculosisthat has never been reported previously. The results suggest thatdexamethasone may deliver clinical effects via mechanisms otherthan generalized immunosuppression, and this has important im-plications for the rationale design of other adjuvant therapies.

Glucocorticoids (GCs), like dexamethasone, continue to be themajor immunomodulatory agents used in clinical medicine today.

FIGURE 6. Activation-induced cell death of cere-brospinal fluid T cells. Shown are the mean percentage(with 25th and 75th percentiles) of viable cerebrospinalfluid or peripheral blood CD3� T cells present after 4 hof in vitro culture in the presence of either mediumalone or a T cell stimulus (phorbol ester and calciumionophore (PMA/IONO)). T cells were isolated from sixTBM patients (three patients on placebo and three pa-tients on dexamethasone) at multiple time points duringthe first 2 wk of therapy. There were significantly fewerviable cerebrospinal fluid CD3� T cells remaining aftera 4-h culture with PMA/IONO (p � 0.05, t test) com-pared with peripheral blood CD3� T cells.

Table II. Concentration of monocyte-derived inflammatory cytokines inwhole-blood cultures stimulated with mycobacterial Agsa

Parameter

Mean Responses 3–14 Days after ReceivingStudy Drugb

pcDexamethasone (n � 18) Placebo (n � 15)

TNF (pg/ml)d 25.87 � 45.64 14.9 � 11.6 NSIL-10 (pg/ml) 39.42 � 47.8 60.1 � 40 NSIL-1� (ng/ml) 0.2 � 0.2 0.1 � 0.1 NSIL-6 (ng/ml) 29 � 34.6 22.2 � 18.2 NSIL-8 (ng/ml) 95.1 � 109.9 74.8 � 43.9 NS

a Paired whole-blood samples were collected from TBM patients on day 0 andagain between days 3 and 14. Blood was stimulated with a lysate of M. tuberculosisH37Rv (5 �g/ml) for 48 h, and the plasma supernatant was analyzed.

b The median time to sample collection was 7 days in the placebo arm and 8 daysin the dexamethasone arm.

c Cytokine concentrations were not significantly different between patients ran-domized to placebo or dexamethasone (Mann-Whitney U test). Values at day 0 in thetwo study arms were also not significantly different (data not shown).

d Values for each cytokine represent the mean concentration (�SD) detected inthe supernatant of blood cultures collected between days 3 and 14 of treatment.

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GCs act by binding to a specific but ubiquitous GC receptor that onactivation translocates to the nucleus. GCs modulate cytokine ex-pression by a combination of genomic mechanisms (reviewed inRefs. 8 and 9). Within the nucleus, the activated GC-receptor com-plex can 1) bind to and inactivate key proinflammatory transcrip-tion factors (e.g., AP-1, NF-�B), which takes place at the promo-tor-responsive elements of these factors but has also been reportedwithout the presence of DNA; 2) via GC-responsive elements, up-regulate the expression of cytokine inhibitory proteins (e.g., I�B,which inactivates the transcription factor NF-�B and thereby thesecondary expression of a series of cytokines); and 3) reduce thehalf-life time of cytokine mRNAs. In studies with triggered humanblood mononuclear cells in culture, GCs strongly diminished theproduction of the initial-phase cytokines IL-1� and TNF-� and theimmunomodulatory cytokines IL-2, IL-3, IL-4, IL-5, IL-10, IL-12,and IFN-�, as well as of IL-6 and IL-8 (10). Conversely, in vivoand in vitro studies have suggested GCs can also have enhancingeffects on the immune response (11–14).

Surprisingly, this study found that dexamethasone did not alterthe kinetics of a range of routine clinical markers of inflammation(CSF-blood glucose ratio, opening pressure, or leukocyte count) ordramatically attenuate cerebrospinal fluid cytokines or chemo-

kines. As described previously in TBM (7, 15–18), the cerebro-spinal fluid contained a rich cytokine milieu; concentrations ofIFN-�, IL-6, IL-10, and IL-8 were high and remained elevated forseveral months. Of note, there was a trend toward decreased ce-rebrospinal fluid IFN-� concentrations in steroid-treated patientsduring the first week of therapy. There was also a trend towarddiminished IFN-� production when whole blood from dexametha-sone-treated patients was stimulated with mitogen but not myco-bacterial Ag. Collectively, these data might suggest a dexametha-sone-mediated effect on IFN-� production; additional studies willbe needed to confirm this association.

Important inflammatory mediators like TNF, IL-12p70, andIL-1� were infrequently detected in acute TBM samples anddropped below detectable levels soon after the initiation of ther-apy. The relevance of these cytokines to host defense and diseasepathogenesis during mycobacterial infection has been revealed re-peatedly in animal models (19–21) but is less certain in humandisease. In particular, TNF has been causally implicated in diseasepathogenesis in a rabbit model of TBM (1). However, cerebrospi-nal fluid TNF concentrations in this study were not measurablyeffected by adjunctive dexamethasone. Similar observations weremade by Donald et al. (16) in children with TBM and randomized

FIGURE 7. T cell responses to mycobacterial Ag or mitogen stimulation measured using whole-blood assays from adult TBM patients randomized todexamethasone or placebo. The data depicts the mean concentration (�95% confidence interval) of IFN-� in plasma supernatants elicited by a 48-hstimulation with a WCL of M. tuberculosis H37Rv (A), rESAT-6 (B), M. tuberculosis H37Rv CFPs (C), or PHA (D). The concentration of IFN-� producedby PBS-stimulated wells was subtracted from Ag-stimulated wells.

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to prednisone; CSF concentrations of TNF, IL-1�, and IFN-� wereunaffected by steroids. Moreover, efforts to suppress TNF expres-sion in vivo using thalidomide have failed to improve outcome inTBM (22). Other inflammatory and vasodilatory mediators, suchas prostaglandins and leukotrienes, may also be relevant to diseasepathogenesis, but were beyond the scope of this study.

Dexamethasone has well-documented effects on monocyte andT cell function in vitro. We therefore expected dexamethasonewould modulate the ex vivo response of monocytes and T cells toantigenic stimulation. However, mycobacterial Ag-triggeredwhole-blood monocyte and T cell assays, plus IFN-� ELISPOTassays, revealed no significant differences in responses betweenpatients in the two treatment arms. These data imply that steroids,in this clinical setting and with the number of samples available foranalysis, do not dramatically attenuate monocyte responses to Agstimulation or Ag-specific, T cell-derived IFN-� responses in vivo.

The phenotypes of cerebrospinal fluid leukocytes in patientswith TBM have not been reported previously, and their role inpathogenesis is unknown. CD3�CD4� T cells were the dominantlymphocyte subset in the cerebrospinal fluid and they coexpressedCD45RA� and CD45RO�, a phenotype identical with alveolarCD4� lymphocytes recovered from the lungs of pulmonary tuber-culosis patients (23). In healthy individuals, the peripheral bloodcontains small numbers of these CD45RA�CD45RO� helper T

cells that primarily produce IFN-� and are in the cell cycle (24,25). Cerebrospinal fluid T cells also expressed the Th1 cell-asso-ciated chemokine receptor CCR5, but not CCR7, a marker of naiveT cells (26). Ligands for CCR5 include RANTES and MCP-1,both of which were present in acute cerebrospinal fluid from TBMpatients. Because a low cerebrospinal fluid white cell count at pre-sentation is independently associated with poor outcome in TBM(6), the recruitment of lymphocytes, such as those described here,to the CNS appears to have a beneficial effect on disease outcome.However, there was no evidence that dexamethasone influencedthe phenotype or kinetics of lymphocytes within the cerebrospinalfluid or blood, although we appreciate that the number of samplesmeasured in this part of the study may have been inadequate todetect significant differences between treatment arms.

How many of the cerebrospinal fluid T cells were specific formycobacterial Ags was difficult to define, because these cells un-derwent rapid activation-induced cell death ex vivo by a mecha-nism unrelated to their expression of FAS (CD95). These obser-vations may preclude the application of in vitro mycobacterial-specific diagnostic tests on cerebrospinal fluid that rely onfunctional T cell responses (e.g., IFN-� production by Ag-stimu-lated T cells in ELISPOT assays) (27).

Dexamethasone did not detectably modulate parameters thathave been independently associated with death in TBM, such as

FIGURE 8. IFN-� ELISPOT responses in PBMCs from adult TBM patients randomized to dexamethasone or placebo after coculture with mycobacterialAgs or peptides. The data depict the mean number (�95% confidence interval) of IFN-� SFU/million PBMCs elicited by stimulation with overlappingpeptides spanning the ESAT-6 protein (A), rESAT-6 (B), and PPD (C). The background frequency of IFN-� SFU elicited by PBS stimulation was subtractedfrom Ag-stimulated wells.

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the cerebrospinal fluid-blood glucose ratio and white cell count (6).However, dexamethasone significantly reduced cerebrospinal fluidtotal protein concentrations in the first week of therapy, consistentwith previous studies in children (5). This effect on cerebrospinalfluid total protein concentration is unlikely to be due to a reductionin the leakage of serum proteins into the cerebrospinal fluid how-ever, because the cerebrospinal fluid-blood albumin index was notsignificantly altered by dexamethasone. Future studies in TBMpatients should aim to define the protein content of cerebrospinalfluid, the factors that influence its production and reabsorption, andtheir relationship to osmotic pressure.

It remains possible that dexamethasone provides clinical benefitin TBM through mechanisms unrelated to inflammation in theCNS. For example, severe adverse drug reactions (e.g., hepatitis)that result in changes or discontinuation of antituberculosis therapyare an independent risk factor for death in TBM patients (6). Be-cause patients randomized to dexamethasone have significantlyfewer severe adverse events during treatment (6), it remains pos-sible that dexamethasone exerts some of its benefits simply byfacilitating continuous antituberculosis therapy.

This study characterized molecular and cellular aspects of TBMpathogenesis and suggests adjunctive dexamethasone does not im-prove survival from TBM by dramatically attenuating prominentmarkers of the immune response in the subarachnoid space or pe-ripheral blood. Other immunological and pathophysiologicalmechanisms may be important; their identification would facilitaterationale drug selection for a disease that still causes significantmortality and morbidity.

AcknowledgmentsWe thank all of the doctors and nurses from the Hospital for TropicalDiseases who cared for the patients.

DisclosuresThe authors have no financial conflict of interest.

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