BACKGROUND
tuberculosis is a major cause of morbidity and mortality worldwide, causing approximately  <dig> millions deaths annually  <cit> .
mycobacterium tuberculosis, the causative agent of tuberculosis is a successful pathogen due to its ability to down regulate host immune responses.
the primary site of infection for m. tuberculosis is the alveolar macrophage.
in cases where pulmonary infection cannot be controlled the organism disseminates via blood to other sites.
pulmonary involvement is seen in the majority of tuberculosis cases however infections of extra-pulmonary sites such as lymph nodes, skeletal, abdominal and genito-urinary sites also remain common  <cit> .
local cellular responses required for restriction of infection with m. tuberculosis are fairly well understood.
granulomas which are the characteristic histopathologic lesions of tuberculosis can be formed in any infected tissue.
granulomas exhibit a characteristic mixture of macrophages and lymphocytes and the outcome of infection depends on the interplay between immune activating cytokines produced at this site.
t cell interferon -gamma  and macrophage activating tumor necrosis factor-alpha  are critical for protection and play a central role in granuloma formation  <cit> .
recent studies have also indicated an important role for chemokines in granuloma formation  <cit> .
chemokines are small mass chemotactic cytokines produced by epithelial cells, mast cells, monocytes and neutrophils.
chemokines can be grouped into structurally different families including c-c chemokines; monocyte chemotactic protein- <dig> , macrophage inflammatory protein-1α , mip-1β  and regulated-upon-activation, normally t-cell-expressed and-secreted  and c-x-c chemokines such as, il- <dig> , mig  and ip- <dig> .
these are all activated by m. tuberculosis  <cit> .
ccl <dig> is the most potent chemoattractant and activator for monocytes and attracts cd <dig> and γδ t cells  <cit> .
ccl <dig> has also been shown to play a role in protection against murine tuberculosis  <cit> .
cxcl <dig> is the most potent attractant and activating factor for neutrophils and is chemotactic for lymphocytes  <cit> .
cxcl <dig> is required for stimulation of a pro-inflammatory response against m. tuberculosis and its components  <cit> .
previous studies have shown that in vivo  serum levels of ifnγ, il- <dig>  <cit> , tnfα  <cit> , cxcl <dig> and il- <dig>  <cit>  are raised in pulmonary tuberculosis.
circulating levels of chemokines cxcl <dig>  ccl <dig> and rantes are raised in bronchoalveolar lavage fluid  and alveolar macrophages from pulmonary tuberculosis patients  <cit> .
increased cxcl <dig> and ccl <dig> activation is also observed in patients with tuberculous pleurisy  <cit>  and meningitis  <cit> .
in addition, the expression levels of cytokines and chemokines have been correlated with disease severity  <cit>  and with subsequent recovery after treatment  <cit> .
we have focused on immune responses in newly diagnosed untreated tuberculosis patients with either pulmonary, or extra-pulmonary disease.
we observed that systemic levels of cxcl <dig> and tnfα were raised in all patients while ccl <dig> was not.
using an ex vivo whole blood assay we observed differential mycobacterium-induced and lps stimulated responses between patients with pulmonary and extra-pulmonary tuberculosis.
mycobacterium-induced ccl <dig> and tnfα, and lps-induced tnfα responses were greater in pulmonary as compared with extra-pulmonary tuberculosis.
however, both mycobacterium-, and lps-induced cxcl <dig> responses were comparable between patient groups.
RESULTS
patient characteristics
hematological characteristics of tuberculosis patients and healthy endemic controls are shown in table  <dig>  median values obtained for pulmonary  and extra-pulmonary tuberculosis  were not different from controls, although for total lymphocyte and neutrophil counts were higher in tuberculosis patients.
the erythrocyte sedimentation rate was raised in both patient groups as compared with controls.
serum ccl <dig>  cxcl <dig> and tnfα in tuberculosis patients
we determined circulating levels of ccl <dig>  cxcl <dig> and tnfα in sera of pulmonary and extra-pulmonary tuberculosis patients and controls .
no difference was observed in serum ccl <dig> levels between the pul-tb, epul-tb and control  groups .
however, serum cxcl <dig> was significantly greater in both pul-tb  and epul-tb  as compared with controls .
serum tnfα was also significantly greater in both pul-tb  and in epul-tb  as compared with controls .
bcg-, and lps -induced ccl <dig>  cxcl <dig> and tnfα
a time course profile of m. bovis bcg – and lps-induced ccl <dig>  cxcl <dig> and tnfα secretion was determined in healthy controls, see fig.
<dig>  bcg infection induced cxcl <dig> secretion within  <dig> –  <dig> h post stimulation , followed by tnfα  and then by ccl <dig> .
lps induced higher levels of all three cytokines as compared with bcg.
in addition, cxcl <dig>  tnf α and ccl <dig> were secreted more rapidly by lps.
peak secretion of chemo/cytokines was observed between  <dig> and  <dig> h post-stimulation, therefore bcg and lps induced responses were subsequently studied at these time intervals.
ccl <dig>  cxcl <dig> and tnfα secretion in whole blood cells from tuberculosis patients
baseline  levels of ccl <dig>  cxcl <dig> and tnfα were determined in pul-tb, epul-tb and control groups.
chemokine activity was measured in supernatants of un-stimulated whole blood cells after  <dig> h and  <dig> h of ex vivo culture.
at  <dig> h, increased levels of ccl <dig> were observed in the pul-tb group;  <dig> pg/ml   as compared with epul-tb;  <dig> pg/ml  and controls;  <dig> pg/ml .
spontaneous secretion of cxcl <dig> after  <dig> h was : pul-tb;  <dig> pg/ml , epul-tb;  <dig> pg/ml  and controls;  <dig> pg/ml .
spontaneous tnfα secretion at  <dig> h was: pul-tb;  <dig> pg/ml , e-pul-tb;  <dig> pg/ml  and controls;  <dig> pg/ml .
there were no significant differences between groups in their baseline levels of ccl <dig>  cxcl <dig> or tnfα .
spontaneous levels of cytokines measured after  <dig> h  of in vitro culture showed a similar trend as observed at  <dig> h, with no difference between groups.
we subsequently investigated lps-induced ccl <dig>  cxcl <dig> and tnfα in tuberculosis patients.
table  <dig> illustrates chemokine activity measured at  <dig> and  <dig> h post lps stimulation.
spontaneous chemokine secretion  have been subtracted from the values indicated in the table.
lps-induced ccl <dig> activation in pul-tb was significantly greater than in epul-tb at  <dig> h  and at  <dig> h  post-stimulation.
lps-induced cxcl <dig> secretion was comparable between patients and endemic controls.
at  <dig> h, tnfα was significantly greater in pul-tb as compared with epul-tb  and also as compared with controls , whilst no difference was observed between these groups at  <dig> h.
bcg-induced chemokine secretion in tuberculosis patients and controls at  <dig> h post-stimulation is illustrated in fig.
<dig>  baseline levels of chemokine secretion have been subtracted and are described in the text above.
bcg- induced ccl <dig> was significantly greater in pulmonary tuberculosis as compared with extra-pulmonary at both  <dig> h   and  <dig> h  post-stimulation.
ccl <dig> secreted in whole blood cells from pulmonary patients was also greater as compared with controls at  <dig> h   and  <dig> h .
there was no difference in bcg-induced cxcl <dig> between pulmonary or extra-pulmonary tuberculosis patients.
similarly, no difference was noted between patients and controls at both time points studied .
in addition, we did not observe any differences in bcg-induced tnfα between controls and tuberculosis patients, p >  <dig>   at either time interval studied.
although the trend of tnfα secretion was lower in the extra-pulmonary group as compared with the pulmonary group, this difference was also not significant.
discussion
we have used an ex vivo whole blood assay to study differences in immune parameters in pulmonary and extra-pulmonary tuberculosis.
we observed that bcg-induced ccl <dig> responses in pulmonary tuberculosis were greater as compared to both extra-pulmonary tuberculosis and control groups.
lps stimulated ccl <dig> and tnfα responses were also raised in pulmonary tuberculosis as compared with extra-pulmonary tuberculosis patients.
we included both ppd skin test positive and negative individuals to be representative of our healthy endemic population, where a large proportion of bcg vaccinated individuals remain non-reactive to the ppd skin test antigen  <cit> .
our tuberculosis patient group was small because of stringent selection criteria.
we excluded most of those patients who had received some form of anti-tuberculous treatment for any length of time.
due to poor therapeutic practices in pakistan the majority of patients presenting to a tertiary care center such as akuh have received variable course anti-tuberculous therapy  <cit> .
there were three times as many female than male patients in our study and this ratio is representative of female preponderance in the disease pattern in pakistan  <cit> .
in congruence with previous studies, neutrophils and monocyte counts were not significantly altered in patient groups  <cit> .
however, erythrocyte sedimentation was significantly higher in tuberculosis patients, reflective of active infection  <cit> .
two extra-pulmonary tuberculosis patients  showed lung involvement on chest x-ray.
these were grouped based on their predominant extra-pulmonary disease site and found that their responses correlated with others in the group.
the raised circulating levels of cxcl <dig> we observe in tuberculosis patients correlate with previous reports  <cit> .
increased cxcl <dig> has been associated with increased neutrophils in broncho-alveolar lavage  <cit>  and pleural fluid  <cit> .
however, we did not find increased neutrophils or monocytes in peripheral circulation of our patients.
we observed raised tnfα in sera of tuberculosis patients.
this is contrast to reports by juffermans et al.
who found that tnfα was not raised in sera of tuberculosis patients  <cit> , while additional reports by olobo et al.
indicate that serum tnfα is increased in healthy contacts  <cit> .
we did not observe any increase in circulating ccl <dig>  correlating with reports by lee et al.
<cit>  who compared sera from tuberculosis patients with healthy tuberculin reactive controls from a disease endemic region.
this was in contrast with juffermans et al.
who observed raised ccl <dig> in sera of tuberculosis patients as compared with healthy individuals from a non-endemic region  <cit> .
ex vivo stimulation of whole blood leucocytes provides useful information on control of cytokine secretion, since it utilizes the host environment  <cit> .
whole blood assays have been shown to provide useful insight into the immunopathology of tuberculosis infection  <cit> .
the time course profiles of ccl <dig>  cxcl <dig> and tnfα secretion we observed in response to bcg and lps corresponded to previous reports of mycobacterium and lps induced activation of purified monocytes and adherent cells  <cit> .
further, after lps stimulation we observed greater cytokine activation than after mycobacterial stimulation as has been shown in the case of cytokines such as cxcl <dig>  <cit> .
therefore the data we obtained using our whole blood assay correlated with patterns observed using purified subsets of cells.
increased ccl <dig> in pulmonary tuberculosis may be related to localization of infection to the lung and may subsequently prevent disease dissemination.
we observed raised ccl <dig> in pulmonary tuberculosis compared with controls in response to bcg stimulation.
raised ccl <dig> activation in tuberculosis correlates with previous studies  <cit> .
however, this contrasts with reports by lee et al.
who did not observe any increase in ccl <dig> secreted by adherent cells from pulmonary tuberculosis patients as compared with healthy tuberculin reactors – in response to ppd or m. tuberculosis  <dig> kd antigen  <cit> .
one difference as compared to our study was that lee et al.
used healthy controls with a ppd skin test induration >  <dig> mm, while our control group comprised of either ppd negative or ppd positive individuals with induration >  <dig> mm.
in addition, the use of live mycobacteria and unprocessed whole blood is likely to give more physiologically relevant results for in vivo chemokine and cytokine activation as compared with recombinant antigen or subunit preparations.
we also observed greater ccl <dig> activation in pulmonary tuberculosis as compared with extra-pulmonary disease in response to both bcg and lps stimulation.
lps-induced cxcl <dig> was not raised in whole blood cells from patients and this was paradoxical with increased levels in sera of these patients.
differences between in vivo  and in vitro  activation have been shown previously  <cit> .
previously, we have shown increased spontaneous cxcl <dig> in disseminated mycobacterial  infections while inducible cxcl <dig> was not raised in patients  <cit> .
therefore, increased levels of cxcl <dig> in sera may be indicative of pathology in these patients and may not contribute to the effective recruitment of cells.
tnfα levels were raised in pulmonary as compared with both extra-pulmonary tuberculosis and controls in response to lps but not bcg stimulation.
this difference in tnfα levels was evident at the earlier  but not later time interval.
probably, due to the reduced time course profile of tnfα by  <dig> h. lack of difference in tnfα in response to bcg between groups, may also be due to reduced overall levels induced by mycobacterium as compared with the lps.
CONCLUSIONS
macrophages and other polymorphonuclear cells play essential roles in initiation and maintenance of immune responses to m. tuberculosis.
pro-inflammatory tnfα, ccl <dig> and cxcl <dig> are important in maintaining a balance between recruitment of other macrophages and also t cells to restrict infection.
ccl <dig> contributes to anti-mycobacterial inflammatory response by attracting monocytes and t lymphocytes.
raised ccl <dig> in pulmonary tuberculosis may lead to greater monocyte mediated immunity.
reduced ccl <dig> concomitant with lowered tnfα in extra-pulmonary tuberculosis may lead to lowered protective cellular responses and increased dissemination in these patients.

 tuberculosis is a major cause of morbidity and mortality worldwide, causing approximately millions deaths annually. mycobacterium tuberculosis, the causative agent of tuberculosis is a successful pathogen due to its ability to down regulate host immune responses. the primary site of infection for m. tuberculosis is the alveolar macrophage. in cases where pulmonary infection cannot be controlled the organism disseminates via blood to other sites. pulmonary involvement is seen in the majority of tuberculosis cases however infections of extra-pulmonary sites such as lymph nodes, skeletal, abdominal and genito-urinary sites also remain common. local cellular responses required for restriction of infection with m. tuberculosis are fairly well understood. granulomas which are the characteristic histopathologic lesions of tuberculosis can be formed in any infected tissue. granulomas exhibit a characteristic mixture of macrophages and lymphocytes and the outcome of infection depends on the interplay between immune activating cytokines produced at this site. t cell interferon -gamma and macrophage activating tumor necrosis factor-alpha are critical for protection and play a central role in granuloma formation. recent studies have also indicated an important role for chemokines in granuloma formation. chemokines are small mass chemotactic cytokines produced by epithelial cells, mast cells, monocytes and neutrophils. chemokines can be grouped into structurally different families including c-c chemokines; monocyte chemotactic protein-, macrophage inflammatory protein-1α, mip-1β and regulated-upon-activation, normally t-cell-expressed and-secreted and c-x-c chemokines such as, il-, mig and ip-. these are all activated by m. tuberculosis. ccl is the most potent chemoattractant and activator for monocytes and attracts cd and γδ t cells. ccl has also been shown to play a role in protection against murine tuberculosis. cxcl is the most potent attractant and activating factor for neutrophils and is chemotactic for lymphocytes. cxcl is required for stimulation of a pro-inflammatory response against m. tuberculosis and its components. previous studies have shown that in vivo serum levels of ifnγ, il-, tnfα, cxcl and il- are raised in pulmonary tuberculosis. circulating levels of chemokines cxcl ccl and rantes are raised in bronchoalveolar lavage fluid and alveolar macrophages from pulmonary tuberculosis patients. increased cxcl and ccl activation is also observed in patients with tuberculous pleurisy and meningitis. in addition, the expression levels of cytokines and chemokines have been correlated with disease severity and with subsequent recovery after treatment. we have focused on immune responses in newly diagnosed untreated tuberculosis patients with either pulmonary, or extra-pulmonary disease. we observed that systemic levels of cxcl and tnfα were raised in all patients while ccl was not. using an ex vivo whole blood assay we observed differential mycobacterium-induced and lps stimulated responses between patients with pulmonary and extra-pulmonary tuberculosis. mycobacterium-induced ccl and tnfα, and lps-induced tnfα responses were greater in pulmonary as compared with extra-pulmonary tuberculosis. however, both mycobacterium-, and lps-induced cxcl responses were comparable between patient groups.  patient characteristics hematological characteristics of tuberculosis patients and healthy endemic controls are shown in table median values obtained for pulmonary and extra-pulmonary tuberculosis were not different from controls, although for total lymphocyte and neutrophil counts were higher in tuberculosis patients. the erythrocyte sedimentation rate was raised in both patient groups as compared with controls. serum ccl cxcl and tnfα in tuberculosis patients we determined circulating levels of ccl cxcl and tnfα in sera of pulmonary and extra-pulmonary tuberculosis patients and controls. no difference was observed in serum ccl levels between the pul-tb, epul-tb and control groups. however, serum cxcl was significantly greater in both pul-tb and epul-tb as compared with controls. serum tnfα was also significantly greater in both pul-tb and in epul-tb as compared with controls. bcg-, and lps -induced ccl cxcl and tnfα a time course profile of m. bovis bcg – and lps-induced ccl cxcl and tnfα secretion was determined in healthy controls, see fig. bcg infection induced cxcl secretion within – h post stimulation, followed by tnfα and then by ccl. lps induced higher levels of all three cytokines as compared with bcg. in addition, cxcl tnf α and ccl were secreted more rapidly by lps. peak secretion of chemo/cytokines was observed between and h post-stimulation, therefore bcg and lps induced responses were subsequently studied at these time intervals. ccl cxcl and tnfα secretion in whole blood cells from tuberculosis patients baseline levels of ccl cxcl and tnfα were determined in pul-tb, epul-tb and control groups. chemokine activity was measured in supernatants of un-stimulated whole blood cells after h and h of ex vivo culture. at h, increased levels of ccl were observed in the pul-tb group; pg/ml as compared with epul-tb; pg/ml and controls; pg/ml. spontaneous secretion of cxcl after h was: pul-tb; pg/ml, epul-tb; pg/ml and controls; pg/ml. spontaneous tnfα secretion at h was: pul-tb; pg/ml, e-pul-tb; pg/ml and controls; pg/ml. there were no significant differences between groups in their baseline levels of ccl cxcl or tnfα. spontaneous levels of cytokines measured after h of in vitro culture showed a similar trend as observed at h, with no difference between groups. we subsequently investigated lps-induced ccl cxcl and tnfα in tuberculosis patients. table illustrates chemokine activity measured at and h post lps stimulation. spontaneous chemokine secretion have been subtracted from the values indicated in the table. lps-induced ccl activation in pul-tb was significantly greater than in epul-tb at h and at h post-stimulation. lps-induced cxcl secretion was comparable between patients and endemic controls. at h, tnfα was significantly greater in pul-tb as compared with epul-tb and also as compared with controls, whilst no difference was observed between these groups at h. bcg-induced chemokine secretion in tuberculosis patients and controls at h post-stimulation is illustrated in fig. baseline levels of chemokine secretion have been subtracted and are described in the text above. bcg- induced ccl was significantly greater in pulmonary tuberculosis as compared with extra-pulmonary at both h and h post-stimulation. ccl secreted in whole blood cells from pulmonary patients was also greater as compared with controls at h and h. there was no difference in bcg-induced cxcl between pulmonary or extra-pulmonary tuberculosis patients. similarly, no difference was noted between patients and controls at both time points studied. in addition, we did not observe any differences in bcg-induced tnfα between controls and tuberculosis patients, p > at either time interval studied. although the trend of tnfα secretion was lower in the extra-pulmonary group as compared with the pulmonary group, this difference was also not significant. discussion we have used an ex vivo whole blood assay to study differences in immune parameters in pulmonary and extra-pulmonary tuberculosis. we observed that bcg-induced ccl responses in pulmonary tuberculosis were greater as compared to both extra-pulmonary tuberculosis and control groups. lps stimulated ccl and tnfα responses were also raised in pulmonary tuberculosis as compared with extra-pulmonary tuberculosis patients. we included both ppd skin test positive and negative individuals to be representative of our healthy endemic population, where a large proportion of bcg vaccinated individuals remain non-reactive to the ppd skin test antigen. our tuberculosis patient group was small because of stringent selection criteria. we excluded most of those patients who had received some form of anti-tuberculous treatment for any length of time. due to poor therapeutic practices in pakistan the majority of patients presenting to a tertiary care center such as akuh have received variable course anti-tuberculous therapy. there were three times as many female than male patients in our study and this ratio is representative of female preponderance in the disease pattern in pakistan. in congruence with previous studies, neutrophils and monocyte counts were not significantly altered in patient groups. however, erythrocyte sedimentation was significantly higher in tuberculosis patients, reflective of active infection. two extra-pulmonary tuberculosis patients showed lung involvement on chest x-ray. these were grouped based on their predominant extra-pulmonary disease site and found that their responses correlated with others in the group. the raised circulating levels of cxcl we observe in tuberculosis patients correlate with previous reports. increased cxcl has been associated with increased neutrophils in broncho-alveolar lavage and pleural fluid. however, we did not find increased neutrophils or monocytes in peripheral circulation of our patients. we observed raised tnfα in sera of tuberculosis patients. this is contrast to reports by juffermans et al. who found that tnfα was not raised in sera of tuberculosis patients, while additional reports by olobo et al. indicate that serum tnfα is increased in healthy contacts. we did not observe any increase in circulating ccl correlating with reports by lee et al. who compared sera from tuberculosis patients with healthy tuberculin reactive controls from a disease endemic region. this was in contrast with juffermans et al. who observed raised ccl in sera of tuberculosis patients as compared with healthy individuals from a non-endemic region. ex vivo stimulation of whole blood leucocytes provides useful information on control of cytokine secretion, since it utilizes the host environment. whole blood assays have been shown to provide useful insight into the immunopathology of tuberculosis infection. the time course profiles of ccl cxcl and tnfα secretion we observed in response to bcg and lps corresponded to previous reports of mycobacterium and lps induced activation of purified monocytes and adherent cells. further, after lps stimulation we observed greater cytokine activation than after mycobacterial stimulation as has been shown in the case of cytokines such as cxcl. therefore the data we obtained using our whole blood assay correlated with patterns observed using purified subsets of cells. increased ccl in pulmonary tuberculosis may be related to localization of infection to the lung and may subsequently prevent disease dissemination. we observed raised ccl in pulmonary tuberculosis compared with controls in response to bcg stimulation. raised ccl activation in tuberculosis correlates with previous studies. however, this contrasts with reports by lee et al. who did not observe any increase in ccl secreted by adherent cells from pulmonary tuberculosis patients as compared with healthy tuberculin reactors – in response to ppd or m. tuberculosis kd antigen. one difference as compared to our study was that lee et al. used healthy controls with a ppd skin test induration > mm, while our control group comprised of either ppd negative or ppd positive individuals with induration > mm. in addition, the use of live mycobacteria and unprocessed whole blood is likely to give more physiologically relevant results for in vivo chemokine and cytokine activation as compared with recombinant antigen or subunit preparations. we also observed greater ccl activation in pulmonary tuberculosis as compared with extra-pulmonary disease in response to both bcg and lps stimulation. lps-induced cxcl was not raised in whole blood cells from patients and this was paradoxical with increased levels in sera of these patients. differences between in vivo and in vitro activation have been shown previously. previously, we have shown increased spontaneous cxcl in disseminated mycobacterial infections while inducible cxcl was not raised in patients. therefore, increased levels of cxcl in sera may be indicative of pathology in these patients and may not contribute to the effective recruitment of cells. tnfα levels were raised in pulmonary as compared with both extra-pulmonary tuberculosis and controls in response to lps but not bcg stimulation. this difference in tnfα levels was evident at the earlier but not later time interval. probably, due to the reduced time course profile of tnfα by h. lack of difference in tnfα in response to bcg between groups, may also be due to reduced overall levels induced by mycobacterium as compared with the lps.  macrophages and other polymorphonuclear cells play essential roles in initiation and maintenance of immune responses to m. tuberculosis. pro-inflammatory tnfα, ccl and cxcl are important in maintaining a balance between recruitment of other macrophages and also t cells to restrict infection. ccl contributes to anti-mycobacterial inflammatory response by attracting monocytes and t lymphocytes. raised ccl in pulmonary tuberculosis may lead to greater monocyte mediated immunity. reduced ccl concomitant with lowered tnfα in extra-pulmonary tuberculosis may lead to lowered protective cellular responses and increased dissemination in these patients. 
m. bovis bcg-induced ex vivo ccl secretion was significantly greater in pulmonary as compared with both extra-pulmonary tuberculosis patients and endemic controls. increased ccl activation in pulmonary tuberculosis may result in a stronger cellular response as compared with extra-pulmonary tuberculosis patients, and this may contribute to the localization of infection to the pulmonary site. we have used an ex vivo assay to investigate differences in immune parameters in patients with either pulmonary or extra-pulmonary tuberculosis. restriction of mycobacterium tuberculosis depends on effective recruitment and subsequent activation of t lymphocytes, mononuclear and polymorphonuclear cells to the site of infection. circulating serum levels of cxcl and tnfα were raised in all tuberculosis patients, while ccl levels were not. whole blood cells were stimulated with non-pathogenic mycobacterium bovis bacille-calmette guerin vaccine strain or bacterial lipopolysaccharide and cyto/chemokines were monitored in supernatants. there was no difference in spontaneous cytokine secretion from whole blood cells between patients and controls. in response to lps stimulation, patients with pulmonary tuberculosis showed increased ccl and tnfα responses as compared with the extra-pulmonary group. serum levels of ccl cxcl and tnfα were measured in patients with pulmonary tuberculosis, extra-pulmonary tuberculosis and bcg-vaccinated healthy volunteers. ccl is essential for recruitment of monocytes and t cells and has been shown to play a role in protection against tuberculosis. ccl is activated by tnfα and is essential for recruitment of monocytes and t cells to the site of mycobacterial infection.
 tuberculosis causes million deaths annually. the most common site of tuberculosis is pulmonary however; extra-pulmonary forms of the disease also remain prevalent. restriction of mycobacterium tuberculosis depends on effective recruitment and subsequent activation of t lymphocytes, mononuclear and polymorphonuclear cells to the site of infection. tumor necrosis factor -α is essential for granuloma formation and is a potent activator of monocyte chemotactic protein. ccl is essential for recruitment of monocytes and t cells and has been shown to play a role in protection against tuberculosis. interleukin - is a potent activator of neutrophils. increased levels of ccl cxcl and tnfα are reported in tuberculosis but their significance in different forms of tuberculosis is as yet unclear. we have used an ex vivo assay to investigate differences in immune parameters in patients with either pulmonary or extra-pulmonary tuberculosis. methods serum levels of ccl cxcl and tnfα were measured in patients with pulmonary tuberculosis, extra-pulmonary tuberculosis and bcg-vaccinated healthy volunteers. whole blood cells were stimulated with non-pathogenic mycobacterium bovis bacille-calmette guerin vaccine strain or bacterial lipopolysaccharide and cyto/chemokines were monitored in supernatants.  circulating serum levels of cxcl and tnfα were raised in all tuberculosis patients, while ccl levels were not. there was no difference in spontaneous cytokine secretion from whole blood cells between patients and controls. m. bovis bcg-induced ex vivo ccl secretion was significantly greater in pulmonary as compared with both extra-pulmonary tuberculosis patients and endemic controls. in response to lps stimulation, patients with pulmonary tuberculosis showed increased ccl and tnfα responses as compared with the extra-pulmonary group. bcg-, and lps-induced cxcl secretion was comparable between patients and controls.  ccl is activated by tnfα and is essential for recruitment of monocytes and t cells to the site of mycobacterial infection. increased ccl activation in pulmonary tuberculosis may result in a stronger cellular response as compared with extra-pulmonary tuberculosis patients, and this may contribute to the localization of infection to the pulmonary site. 