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7/31/2019 TNF Influences Chemokine Expression of Macrophages in Vitro and That of CD11b+ Cells in Vivo During Mycobact
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TNF Influences Chemokine Expression of Macrophages In
Vitro and That of CD11b Cells In Vivo during
Mycobacterium tuberculosis Infection1
Holly M. Scott Algood,* Philana Ling Lin, David Yankura,* Alvin Jones,* John Chan, and
JoAnne L. Flynn2*
Granulomas, focal accumulations of immune cells, form in the lung during Mycobacterium tuberculosis infection. Chemokines,
chemotactic cytokines, are logical candidates for inducing migration of T lymphocytes and monocytes to and within the lung. TNF
influences chemokine expression in some models. TNF-deficient mice infected with M. tuberculosis are highly susceptible to disease,
and granuloma formation is inhibited. Through in vitro assays, we demonstrate that neutralization of TNF in M. tuberculosis-
infected macrophages led to a reduction in many inflammatory chemokines, such as C-C chemokine ligand 5, CXC ligand 9
(CXCL9), and CXCL10. In TNF-deficient mice, immune cells migrated to the lungs early after infection, but did not organize to
form granulomas within the lung. Although chemokine expression, as measured in whole lung tissue, was not different, the
expression of chemokines in the CD11b subset of cells isolated ex vivo from the lungs of TNF-deficient mice had reduced
expression of C-C chemokine ligand 5, CXCL9, and CXCL10 at early time points after TNF neutralization. Local expression of
CXCR3-binding chemokines within the lungs, as determined by in situ hybridization, was also affected by TNF. Therefore, TNF
affects the expression of chemokines by macrophages in vitro and CD11b cells in vivo, which probably influences the local
chemokine gradients and granuloma formation. The Journal of Immunology, 2004, 172: 68466857.
The hallmark of Mycobacterium tuberculosis infection is
granuloma formation. The granuloma is a focal accumu-
lation of immune cells, including macrophages, lympho-
cytes, and, at times, neutrophils (1). Within the granuloma, effector
and bystander cells need to communicate in a coordinated manner.
Signals required from CD4 and CD8 lymphocytes include IFN-
and TNF, which activate macrophages harboring M. tuberculosis
(reviewed in Ref. 2). Activated macrophages have enhanced reac-
tive nitrogen intermediate production and phagolysosome fusion,both of which can lead to destruction of M. tuberculosis bacilli. In
addition, CD8 cytotoxic lymphocytes can kill the bacillus directly
through granulysin-dependent mechanisms or kill the macro-
phages harboring the bacilli (reviewed in Ref. 3).
In recent years, the use of anti-inflammatory drugs targeting
TNF, such as Infliximab and Enbrel, for treatment of chronic in-
flammatory diseases, has highlighted the importance of TNF in
controlling M. tuberculosis, because there is an increased risk of
reactivation tuberculosis in patients treated with these drugs (4, 5).
TNF has many potential effects within the lungs of an infected
individual. In vitro, TNF has been reported to affect apoptosis of
infected macrophages (6, 7), influence secondary signaling to mac-
rophages for NO production (reviewed in Ref. 8), and regulate the
activity of other inflammatory cytokines (9). In vivo, pathology,
control of bacterial numbers, and chemokine expression (in the
liver) have been shown to be affected by TNF (10, 11). The role of
TNF within the lungs in chemokine expression after M. tubercu-
losis infection has not been explored.
Neutralization of TNF in chronically infected mice led to severe
pathology with the loss of granuloma structure (12, 13). Bacterial
loads in the TNF-neutralized, chronically infected mice increased
initially, but stabilized at a level below that generally considered
fatal in C57BL/6 mice. Nonetheless, TNF-neutralized mice suc-
cumbed quickly. Granuloma structural deficiencies and uncon-
trolled infection in mice lacking TNF have been reported by our
group and others, in both acute and chronic infection (10, 1215);
however, the mechanisms by which TNF controls granuloma de-
velopment and maintenance have yet to be elucidated. Studies in
the liver of M. smegmatis and M. tuberculosis i.v.-infected mice
showed that gene expression of a subset of chemokines was re-
duced in TNF-deficient mice (11), but these studies did not reveal
the localized chemokine expression patterns in the lungs, the pri-
mary site of M. tuberculosis infection and control after aerosol
infection.
We hypothesized that TNF influences macrophage chemokine
expression and that these localized differences in expression lead
to changes in granuloma formation. Our results described in this
study demonstrate for the first time that TNF affects chemokine
gene and protein expression by M. tuberculosis-infected macro-
phages both in vivo and in vitro. We also demonstrate that during
the early phases of acute infection, TNF-deficient mice have sim-
ilar numbers and types of immune cells migrating to the lungs,
*Department of Molecular Genetics and Biochemistry and Molecular Virology andMicrobiology Graduate Program, University of Pittsburgh School of Medicine, Pitts-burgh, PA 15261; Childrens Hospital of Pittsburgh, University of Pittsburgh School
of Medicine, Pittsburgh, PA 15213; and
Departments of Medicine and Microbiologyand Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
Received for publication December 18, 2003. Accepted for publication March22, 2004.
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 National Institutes of Health Grant HL71241 (to J.C.and J.L.F.), American Lung Association Grant CI-016-N, a Western PennsylvaniaLung Association Dissertation grant (to H.M.S.), National Institutes of Health/Na-tional Institute of Allergy and Infectious Diseases Grant T32AI49820 (to H.M.S.),and the Bayer/Harold Neu Postdoctoral Fellowship from Infectious Disease Societyof America (to P.L.L.).
2 Address correspondence and reprint requests to Dr. JoAnne L. Flynn, Depart-ment of Molecular Genetics and Biochemistry, University of Pittsburgh Schoolof Medicine, W1111 Biomedical Science Tower, Pittsburgh, PA 15261. E-mailaddress: [email protected]
The Journal of Immunology
Copyright 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00
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indicating that the lack of granuloma formation is a result of a
migration defect within the lung tissue, rather than a defect in the
migration of cells to the lungs. These data support the idea that
TNF affects the expression of many chemokines at the level of
macrophage gene expression, suggesting that differences in local-
ized chemokine gradients may result in disorganized cellular in-
filtrate within the lungs.
Materials and MethodsAnimals
C57BL/6 female mice (Charles River, Rockland, MA) and TNF receptor(TNFR)3p55/ (breeding pairs from Dr. T. Mak, Ontario Cancer Institute,
Toronto, Canada) (16), 8 14 wk old, were used in all experiments. TN-FRp55/ mice were bred in a specific pathogen-free facility at Universityof Pittsburgh. All infected mice were maintained in the BSL3 animal lab-
oratories and routinely monitored for murine pathogens by means of se-rological and histological examinations. The university institutional animal
care and use committee approved all animal protocols used in this study.
Chemical and reagents
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unlessotherwise noted. Middlebrook 7H9 liquid medium and 7H10 agar wereobtained from Difco (Detroit, MI). Abs used in flow cytometric analyses
were obtained from BD PharMingen (San Diego, CA). The cell lineMP6XT-22 (anti-TNF) was obtained from DNAX (Palo Alto, CA) and
used to generate ascites (Harlan Bioproducts, Indianapolis, IN). Abs werepurified endotoxin-free from ascites fluid as previously described (12), andmice were treated with Ab i.v. every 3 days (0.5 mg/dose) starting 4 mo
postinfection. Control mice received normal rat IgG (Jackson Immuno-Research Laboratories, West Grove, PA) with the same dosing regimen. Incell culture MP6XT-22 was used at a concentration of 0.1 mg/ml.
Mycobacteria and infection of mice
M. tuberculosis strain Erdman (Trudeau Institute, Saranac Lake, NY) was
used to infect mice via aerosol (50 100 CFU) as previously described (17).In acute experiments in which a higher inoculum was used, the concen-
tration of M. tuberculosis in the nebulizer was increased, and day 1 CFUwere determined in the lungs. The tissue bacillary load was quanti fied byplating serial dilutions of the tissue homogenates onto 7H10 agar as de-
scribed previously (18). In all chronic M. tuberculosis experiments, infec-tions were performed using the low dose (50 100 CFU) aerosol infection,and mice were used at 4 mo postinfection.
Bone marrow-derived macrophages
Macrophages were derived from C57BL/6 bone marrow based on adher-
ence, as previously described (19). On day 6 of culture macrophages wereinfected with M. tuberculosis (multiplicity of infection, 4). Four hours
postinfection the supernatant was removed, cells were washed, and freshmedium was added.
Flow cytometric analysis of lung cells
To determine cellular infiltrate in the lung, at 10-day intervals lungs wereremoved for flow cytometric analysis. Lungs were subjected to a short
period (20 min) of digestion with 1 mg/ml collagenase A and 25 U/mlDNase (Roche, Mannheim, Germany) at 37C. The suspension was pushedthrough a cell strainer as previously described (20). RBC were lysed with
RBC lysis buffer (NH4Cl/Tris solution), and the single-cell suspension wascounted. The samples were stained with anti-CD4, anti-CD8, anti-CD69, or
anti-Gr1 and anti-CD11b in FACS buffer as previously described (20). Thecells were fixed in 4% paraformaldehyde and collected on a FACSCaliber(BD Biosciences, Mountain View, CA). Analysis was performed using
CellQuest software (BD PharMingen, San Diego, CA).
Histopathology
Tissue samples for histological studies were fixed in 10% normal bufferedformalin, followed by paraffin embedment. For histopathological studies,5- to 6-m sections were stained with Harris H&E.
Ex vivo CD11b isolation
To isolate CD11b cells from the lungs of C57BL/6 mice, a MACS col-umn isolation protocol (Miltenyi Biotech, Auburn, CA) was followed. The
lungs were removed, rinsed in T cell medium, and pushed through a 70-mpore size cell strainer (Fisher, Pittsburgh, PA) using the end of a 5-mlsyringe. After RBC lysis and washing, the cells were labeled with CD11b
microbeads in MACS buffer (2 mM EDTA and 0.5% SDS in PBS) for 15min at 8C. The cells were washed with MACS buffer and then resus-pended in 0.5 ml of buffer. The cells were poured over a 40-m pore sizecell strainer and then applied to a prewashed MS column. After initial
flow-through, the column was washed three times with 0.5 ml of buffer.The column was removed from the magnet, and CD11b cells were elutedfrom the column with 1 ml of buffer. The cells were spun down and re-suspended in 1 ml of TRIzol, and the RNA was isolated.
RNA isolation
RNA was isolated from the lung or cells using the TRIzol isolation pro-tocol with modifications. The lung was homogenized in 3 ml (cells werelysed in 1 ml of TRIzol/2 106 cells) of TRIzol reagent, and then twochloroform extractions were performed. After an isopropanol precipitation,the RNA was washed with 70% ethanol and treated with RNase inhibitor
(Applied Biosystems, Foster City, CA) for 45 min. After treatment at 65Cfor 15 min (to fully resuspend the RNA), the RNA was cleaned, and DNase
was digested using the Qiagen RNA isolation kit, as directed by the man-ufacturer (Qiagen, Valencia, CA).
Real-time RT-PCR
The RNA was reverse transcribed using Superscript II enzyme, as directedby the manufacturer (Invitrogen, Carlsbad, CA). For real-time RT-PCR weused the relative gene expression method (21). hypoxanthine phosphori-
bosyltransferase served as the normalizer, and uninfected lung or macro-phages served as the calibrator. Each primer and probe set was tested forefficiency (results efficient 97% for all primer/probe sets). All sampleswere run in triplicate and with no-reverse transcriptase controls on an ABIPRISM Sequence Detector 7700. Relative gene expression was calculated
as 2( cycle threshold (Ct)), where Ct Ct (gene of interest) Ct (nor-malizer) and the Ct Ct (sample) Ct (calibrator). Results areexpressed as relative gene expression to uninfected samples. The primer
and probe concentrations were used as suggested by Applied Biosystems,with the final concentration of each primer at 400 nM and that of probe at250 nM. The primers (Life Technologies, Grand Island, NY) and probes
(Applied Biosystems) used in this assay were designed using Applied Bio-systems Primer Express software and the sequences are in Table I.
Protein determination
Immunoassay. The quantities of CXCL9 and CXCL10 in the macrophage
culture supernatants were determined with the Quantikine mouse CXCL9and CXCL10 immunoassays (R&D Systems, Minneapolis, MN). These
kits were used exactly as suggested by the manufacturer. The readout wasperformed using the Emax precision microplate reader (Molecular Devices,Sunnyvale, CA).
SearchLight proteome arrays. To determine the protein concentrations ofTNF, C-C chemokine ligand 2 (CCL2), CCL3, CCL5, CCL12, and
CXCL2, a multiplex sample testing service was used. Supernatants werecollected from uninfected and infected macrophages using a pipette. These
supernatants were then were filter-sterilized using an Acrodisc 13-mm sy-
ringe filter with a 0.2-m HT Tuffryn membrane (Pall Corp., Ann Arbor,MI) attached to a 5-ml syringe. The supernatants were stored in 0.5-mlaliquots at 20C. An aliquot was sent to Pierce (Rockford, IL) on dry ice(Boston, MA) for quantitative measurement using multiplexed sandwich
ELISAs.
In situ hybridization.
Cloning. The pGEM-T Easy Vector System (Promega, Madison, WI) wasused to clone CCL5, CXCL9, and CXCL10 PCR products. Primers weredesigned to amplify CCL5, CXCL9, and CXCL10 with restriction enzyme
sites specific for the pGEM-T vector cloning sites. After amplification, theproduct was purified using the QIAquick Nucleotide Removal Kit (Qiagen,Valencia, CA). The ends were digested, and the product was ligated to cutpGEM-T vectors. Competent Escherichia coli (JM109) was transformedwith the ligation reactions, using the suggested protocol (Promega, Mad-
ison, WI). Transformed bacteria were selected based on resistance to am-
picillin and interruption of the lacZ promoter, and the selected colonieswere grown up in LBAMP liquid medium. The plasmid DNA was puri-
fied from this culture by the Qiagen Midiprep protocol (Qiagen). After
3 Abbreviations used in this paper: TNFR, TNF receptor; CCL, C-C chemokine ligand;CXCL, CXC chemokine ligand; LCM, laser capture microscopy; WT, wild type; Ct,
cycle threshold.
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verification of the insert, the plasmids were linearized and used to generateprobes.
Probe generation. Labeled RNA probes were synthesized by in vitro tran-
scription of DNA cloned downstream of T7 or SP6 RNA polymerase in thepGEM-T vector using [35S]UTP as substrate. The MAXIscript In Vitro
Transcription Kit (Ambion, Austin, TX) was used for this protocol, withslight modification. Probes were stored at 80C.
Tissue and slide preparation. Lungs were fixed in 4% paraformaldehydeat 40C for 5 h, washed twice overnight with PBS, followed by a sucrosegradient, and then snap-frozen and stored at 80C. Cryosections (14 m)were cut, and slides were fixed in 4% paraformaldehyde for 20 min, de-hydrated in an ethanol gradient (70% ethanol for 20 min, 80% ethanol for
5 min, then 95% ethanol for 5 min). After air-drying the slides, Ag retrievalwas performed in 0.01 M sodium citrate, with warming in the microwave.After the slides were cooled, the tissues were acetylated twice in 0.25%
acetic anhydride-0.1 M triethanolamine solution. After treatment in anotherethanol dehydration gradient, the slides were air-dried.
Hybridization and washes. Tissue was hybridized to the 35S-labeledprobes as previously described (22). In short, 5 104 cpm/l sense or
antisense probe was hybridized to the tissue under siliconized coverslips(in 1 hybridization buffer containing 10% dextran sulfate formamide, 0.6
M NaCl, yeast tRNA (100 g/ml), and 0.4 M DTT) overnight at 50C.Coverslips were removed, and slides were washed in a series of SSC buff-ers: 5 SSC and 0.1 mM DDT at 42C for 30 min, 2 SSC/50% form-
amide and 0.1 mM DTT at 60C for 20 min, ribowash solution (0.1 M Tris,0.05 M EDTA, and 0.4 M NaCl) at 37 C for 10 min, ribowash solution at37C for 10 min, ribowash solution and 6.25 U of an RNase A T1 mix at37C for 30 min, ribowash solution at 37C for 10 min, 2 SSC at 37Cfor 10 min, and 0.2 SSC at 37C for 10 min. Slides were then dehydratedin 0.3 M ammonium acetate and an ethanol gradient. After the slides werecompletely dried, they were dipped in prewarmed radiography emulsion(Eastman Kodak, Rochester, NY) and dried. Slides were wrapped in a dark
box for 1 wk. Development was performed in D19 developer, followed by
fixation in Rapid Fix. Slides were counterstained in hematoxylin.
Statistical analysis
Three or four mice per group per time point were used for all studies.Statistical analysis was performed on the data using PRISM software for an
unpaired ttest. For bacterial numbers and cell numbers, log transformation
was performed before statistical analysis to normalize the data. A value ofp 0.05 was considered significant.
ResultsSusceptibility to tuberculosis without TNF
Mice deficient in TNF have increased susceptibility to M. tuber-
culosis infection (reviewed in Ref. 23). After low dose aerosol
infection, bacterial burdens in TNF-neutralized mice (MP6-XT22
treated) and TNFRp55/ mice began to diverge from wild-type
(WT) levels by 14 days postinfection, and the mice had signi fi-
cantly higher bacterial burdens than control mice by 18 21 days
postinfection (Fig. 1A and data not shown). The TNF-deficient
mice succumbed by 28 days postinfection.
After TNF neutralization of chronically infected mice, the bac-
terial burden increased by 8 days postinfection (Fig. 1B), and dif-
ferences were significant by 18 days postinfection. We previously
reported that the mean survival time for chronically infected TNF-
neutralized mice was 44 days when infection was initiated by i.v.
infection with moderate doses of M. tuberculosis (12). However,
using low dose inoculum delivered via aerosol to set up the chronic
infection, neutralization of TNF resulted in faster progression of
disease, with a mean survival time of 21 days after initiation of Ab
treatment (Fig. 1C). In the following experiments, we analyzed the
effects of TNF deficiency in acute and chronic models of infection,
using aerosol delivery for all studies.
Cell infiltration during acute infection
During acute mycobacterial infections, TNF-deficient mice do not
form proper granulomas (14, 15, 24). The cell populations migrat-
ing into the lungs were assessed by flow cytometry. No defects in
the appearance of T lymphocytes and macrophages in the lungs
early after infection were observed in TNF-deficient mice com-
pared with controls (Fig. 2). By 21 days postinfection, when bac-
terial burdens were already significantly higher in anti-TNF Ab-
treated mice, the number of neutrophils was significantly higher.
The increased neutrophilic infiltrate was probably a result of the
increased mycobacterial burden in these mice. Histological anal-
ysis of the lung infiltrate indicated that although the cells were
migrating into the lungs, they were not forming organized granu-
loma structures, as previously reported (Refs. 14 and 15 and data
not shown). These data indicate that TNF is not required for early
migration of cells into the lung after infection, but, rather, may
influence migration within the lung.
Cell populations in the lung after anti-TNF treatment in
chronically infected mice
Similar results were obtained when analyzing TNF-neutralized,
chronically infected mice. The cell populations in the lungs of
anti-TNF Ab-treated and IgG control mice were not significantly
different in the anti-TNF Ab-treated mice (Fig. 3). Although there
was a trend toward increasing cell numbers in the anti-TNF-treated
mice, there was variability among the mice during the chronic
stage of infection (see error bars for day 0 time points, Fig. 3), and
mice in the TNF-neutralized group succumbed to reactivation be-
tween days 12 and 25 after Ab treatment (Fig. 1C). There was no
statistical change in the numbers of cells in the lungs compared
with the number of cells on day 0 of Ab treatment.
Histological changes in granuloma structure were observed as
early as 9 days after anti-TNF Ab treatment (representative sec-
tions are shown in Fig. 4). By 20 days of Ab treatment all surviv-
ing anti-TNF treated mice had diffuse cellular infiltrate in their
lungs accompanied by edema within alveolar spaces (Fig. 4).
Table I. Primers and probes used in the TaqMan real-time RT-PCR assaya
Gene Forward Primer (5 to 3) Reverse Primer (5 to 3) TaqMan Probe (5 6-FAM, 3 TAMRA labeled)
CXCL9 TGG AGC AGT GTG GAG TTC GA TCG GCT GGT GCT GAT GC CCC TAG TGA TAA GGA ATG CAC GAT GCT CC
CXCL10 AGA GCA GCA CTT GGG TTC ACG GCA GCA CTT GGG TTC CGG AAG CCT CCC CAT CAG CAC C
CXCL11 GGG CGC TGT CTT TGC ATC AAG CTT TCT CGA TCT CTG CCA T CCC CGG GAT GAA AGC CGT CAA
CCL2 CTT CCT CCA CCA CCA TGC A CCA GCC GGC AAC TGT GA CCC TGT CAT GCT TCT GGG CCT GC
CCL3 ACC AAG TCT TCT CAG CGC CAT TTC CGG CTG TAG GAG AAG CA TGG AGC TGA CAC CCC GAC TGC C
CCL4 CCC GAG CAA CAC CAT GAA G AGA AGG CAG CCA CGA GCA TCT GCG TGT CTG CCC TCT CTC TCC TC
CCL5 CTG CAG CCG CCC TCT G GAC TGC AAG ATT GGA GCA CTT G CTC CCT GCT CGT TTG CCT ACC TCT CC
CCL12 GGA GGA T CA CAA G CA GCC A GT TCA G CA CAG A TC TCC T TA TCC A GT AT TCC CCG GGA AGC TGT GAT CTT CAG
HPRT TTA CCT CAC TGC TTT CCG GAG AAA AGC GGT CTG AGG AGG AAG TAG CAC CTC CTC CGC C
TNF TGA TCC GAG ACG TGG AA ACC GCC TGG AGT TCT GGA A TGG CAG AAG AGG CAC TCC CCC AA
a Primers and probes were designed using Primer Express software. Sequences used for primer and probe synthesis are found in the table.
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These data indicate that TNF plays a direct or indirect role in
controlling migration of immune cells within the lungs and main-
taining granuloma structure. A possible mechanism for control of
migration of cells is through effects on chemokine expression.
TNF affects chemokine expression in M. tuberculosis-infected
macrophages
The effects of TNF on M. tuberculosis-induced chemokine expres-
sion by macrophages were first examined in vitro. After infection
of bone marrow-derived macrophages with M. tuberculosis, the
expression of many inducible C-C and C-X-C chemokines in-
creased relative to that of uninfected macrophages, and mRNA
levels peaked between 4 and 12 h postinfection depending on the
gene (Fig. 5 and data not shown). RNA expression of CXCL10,
CCL12, CCL2, and CCL4 peaked at 4 h, whereas expression of
CXCL9, CXCL11, CCL3, and CCL5 did not peak until 12 h
postinfection. Although RNA expression decreased rapidly after
12 h of infection, the levels remained elevated compared with
those of uninfected macrophages.
As early as 4 h postinfection, TNF expression was induced (Fig.
5). Anti-TNF Ab treatment at the time of infection reduced theexpression of many chemokines (Fig. 5), indicating that optimal
expression of these chemokines was dependent on TNF. The ex-
pression of some chemokines, such as CCL5, was sustained at a
higher level as infection progressed in vitro, and in the presence of
anti-TNF Ab, the expression of CCL5 was reduced 30 70% (de-
pending on the time point analyzed). CCL2 expression was re-
duced by TNF neutralization only at the 4 h point (Fig. 5). TNF
neutralization modestly reduced CCL3 expression early in infec-
tion, but high levels of CCL3 mRNA were sustained in the anti-
TNF Ab-treated, but not control, macrophage cultures (Fig. 5).
CCL12 also had reduced expression in TNF-neutralized cultures
early after infection, but this was up-regulated in the presence of
anti-TNF Ab by 24 h postinfection and decreased again by 48 h
(data not shown).
In a separate set of experiments, anti-TNF Ab was added 4 h
after infection (when unincorporated bacteria were removed from
the culture); the expression of chemokines was examined 24 and
48 h after infection (data not shown). The mRNA levels of CCL5,
CXCL10, and CXCL11 were still significantly reduced at 24 and
48 h postinfection (60, 58, and 84%, respectively). The expression
of CCL12 was significantly increased (data not shown). Treatment
of infected macrophages with isotype control Ab did not alter che-
mokine expression (data not shown). Similar results were obtained
using TNFRp55/ macrophages (data not shown). Addition of
recombinant TNF to the infected macrophage cultures did not lead
to additional chemokine expression (data not shown), suggesting
that the TNF induced by M. tuberculosis infection was sufficient to
affect the expression of the chemokine genes. These data indicated
that TNF induced the expression of CCL5, CXCL9, CXCL10, and
CXCL11, but down-regulated the expression of CCL12.
Chemokine protein production in M. tuberculosis-infected
macrophages
There are little data about post-transcriptional regulation of che-
mokine expression (25, 26). To confirm the gene expression re-
sults, we quantified protein production. Supernatants collected
from uninfected and infected macrophages at 0, 4, 8, 12, 24, and
48 h postinfection (from the cultures used to make RNA in Fig. 5)were used in the immunoassay for CXCL9 and CXCL10 and were
used for multiplex sample testing to quantify CCL2, CCL3, CCL5,
CCL12, CXCL8, and TNF (Fig. 6).
Protein expression of CXCL9 was reduced 30 70% in the pres-
ence of anti-TNF Ab compared with control macrophages, depend-
ing on the time point. The expression of CXCL10 was reduced
20 30%, that of CCL5 was reduced 50 90%, that of CCL3 was
reduced 10 90%, and that of CXCL8 was reduced 25%70% (data
not shown). The expression of CCL2 and CCL12 (data not shown)
followed a similar pattern; in the presence of anti-TNF Ab, ex-
pression was reduced initially, then increased, but by 48 h postin-
fection there was clearly a reduction in the amount of protein
present.
Protein analysis of supernatants from cultures to which anti-TNF
Ab was added at 4 h postinfection confirmed the mRNA macrophage
FIGURE 1. Deficiencies in TNF signaling led to rapid disease progres-
sion. A, CFU in lungs of MP6XT-22 treated mice () were significantly
higher than those in IgG-treated mice (F) by day 18 after aerosol infection
( , p 0.0420; day 21, , p 0.0097; day 25, , p 0.0426). B,
MP6XT-22 treatment of C57BL/6 WT mice 4 mo postinfection led to an
increase in bacterial numbers in the lungs ( , p 0.04). , MP6XT-22-
treated mice; , IgG-treated mice. C, MP6XT-22 treatment led to rapid
reactivation of disease, and mice began to succumb to infection only 15 days
after anti-TNF Ab treatment. The mean survival time for MP6XT-22-treated
mice (when treatment began 4 mo after aerosol infection) was 22 days. Each
time point represents the mean of four mice. The graphs shown are represen-
tative of data from one of four independent experiments.
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data, in that CCL2, CCL5, CCL12, CXCL9, and CXCL10 were in-
duced byM. tuberculosis infection, and CXCL9, CXCL10, and CCL5
were down-regulated in the presence of anti-TNF Ab (data not
shown). In contrast, although there was no difference in the expression
of CCL2 mRNA in anti-TNF-treated macrophages, there was a sig-
nificant reduction in the amount of protein.
Effects of TNF on chemokine expression by pulmonary
macrophages in vivo
Based on the results from macrophages and the findings in thespleen and liver of infected mice from a previous study (11), we
examined the expression of chemokines in the whole infected lung
by both RNase protection assay and real-time RT-PCR. We ex-
pected to observe differences similar to our findings in the in vitro
macrophage experiments, but after low dose aerosol infection, the
induction of chemokines over uninfected lung was not detectable
until 1012 days postinfection. In the aerosol infection model
used, only 50 100 CFU were deposited in the lungs, and only a
small percentage of macrophages were infected, leading to very
low level chemokine expression. There is very little time between
the detectable induction of expression in the whole lung and
increasing bacterial growth in TNF-deficient mice, complicat-
ing analysis of the role of TNF in control of chemokine
expression.
Gene expression measured in whole lung tissue might not ac-curately reflect the relevant gene expression within the granuloma
(27) due to the local nature of the immune response within the
granuloma. For this reason, chemokine expression was measured
from RNA obtained specifically from granulomatous tissue pro-
cured by laser capture microscopy (LCM). Lung sections from
FIGURE 2. Immune cells migrate
into the lungs in MP6XT-22-treated
mice during acute infection. Flow cy-
tometric analysis of the lung was
used to quantify the numbers of
CD4 and CD8 lymphocytes, mac-
rophages (CD11b
Gr1
), and neu-trophils (CD11bGr1) present in
the lungs after aerosol infection. At
21 days postinfection there was a
statistical difference in the number of
macrophages present in the lungs ( ,
p 0.023). There was also a signif-
icantly higher number of neutrophils
on day 14 ( , p 0.0202), day 18
( , p 0.0001), and day 21 ( ,
p 0.0004). Each time point repre-
s e nt s t hr ee o r f ou r m ic e . T he s e
graphs are representative of two in-
dependent experiments.
FIGURE 3. The number of cells
migrating into the lungs did not signif-
icantly change with MP6XT-22 or IgG
treatment of chronically infected mice.
The number of CD4 lymphocytes,
CD8 lymphocytes, macrophages, and
neutrophils in the lungs of MP6-treated
() and IgG-treated () mice did not
significantly change compared with the
numbers of cells present on day 0 of
Ab treatment. Each time point repre-
sents four mice per group. This exper-
iment was repeated twice with similar
results.
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early time points after MP6 or IgG treatment were chosen to assess
the effects of TNF neutralization during a time where bacterial
numbers remained similarin both groups. Differences between anti-
TNF Ab-treated mice and control mice were not evident using
LCM (data not shown). This may be due to collecting many cell
types from the granulomas including T lymphocytes, B lympho-cytes, macrophages, and neutrophils.
Macrophages are a major reservoir for M. tuberculosis and prob-
ably play a key role in orchestrating granuloma formation. As a more
sensitive technique for assessing the effect of TNF on macrophage
chemokine expression, we enriched for macrophages by isolating
CD11b cells from the lungs of acutely infected mice at various time
points after low dose aerosol infection. RNA was isolated from the
CD11b population, and real-time RT-PCR was performed. On day
10 postinfection, the CD11b population of cells was 50% F4/80
and 46% Gr1 after MACS column isolation, indicating that this
technique purified both macrophages and neutrophils from the lungs.
Using this method, induction of chemokines in CD11b cells was still
difficult to detect before 9 days postinfection; at this time point there
was reduced relative expression of chemokines in the TNF-deficient
mice compared with WT, but the expression was very low (data not
shown). Just 3 days later (day 12) chemokine expression rose
dramatically, and the TNF-deficient mice had higher expression
of many chemokines (data not shown).
The aerosol inoculum was increased 20-fold to increase the
number of macrophages initially infected in the lungs, and induc-
tion of chemokines in the macrophage population was detected asearly as 4 days postinfection. There were differences between TN-
FRp55/ and WT mice in the expression of CXCL9, CXCL10,
CCL3, CCL4, CCL5, and CCL12 (Fig. 7A). By 7 days postinfec-
tion the bacterial burdens in the TNFRp55/ mice were signifi-
cantly higher at this dose (data not shown). There were still re-
duced levels of expression of CXCL9, CXCL10, and CCL4, but
now there was increased expression of CCL5 and CCL12 in TN-
FRp55/ mice compared with that in WT mice (Fig. 7A). By day
10 the expression was 10-fold higher than on day 7, and the dif-
ferences between the mouse strains were no longer evident (data
not shown).
CD11b cells were also isolated ex vivo from the lungs of M.
tuberculosis chronically infected mice (treated with IgG or anti-
TNF Ab), and real-time RT-PCR was performed on isolated RNA.
The expressions of CCL3, CCL4, CCL5, CXCL9, and CXCL10
FIGURE 4. Neutralization of TNF in chronically in-
fected mice leads to severe lung pathology. MP6XT-22
treatment of mice led to disorganized cellular infiltra-
tion and fluid in the alveolar spaces of the lungs by 12
days after Ab treatment. IgG-treated mice still exhibited
organized granuloma formation, with clusters of lym-
phocytes and macrophages throughout treatment. Mag-
nification, 100. Sections shown in this figure are rep-resentative of four mice per group. This experiment was
repeated four times with similar results.
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were transiently reduced in CD11b cells isolated from chronically
infected MP6-treated mice (Fig. 7B). The expression of these genes
increased as bacterial burdens increased, and by 12 days postinfection,
when the bacterial burdens in MP6-treated mice were substantially
higher than those in IgG-treated mice, chemokine mRNA levels were
higher than those in control mice.
FIGURE 6. Chemokine protein expression
in M. tuberculosis-infected macrophages was
affected by anti-TNF Ab treatment. Superna-
tants from macrophages cultures infected for
0, 4, 8, 12, 24, and 48 h were collected to
quantitate protein expression after anti-TNF
Ab treatment (or no treatment) of macro-
phages. Supernatants are from the cultures
used for RNA preparations in Fig. 5. ELISAs
were performed for CXCL9 and CXCL10,
whereas protein concentrations for the other
chemokines and TNF were determined by
multiplex protein array. This experiment was
performed twice with similar results.
FIGURE 5. Macrophage expression
of chemokines is partially dependent on
TNF. Real-time RT-PCR was per-
formed on RNA isolated from macro-
phages infected with M. tuberculosis at
0, 4, 8, 12, 24, and 48 h postinfection,
with or without addition of anti-TNF
Ab. The expression levels reported are
relative to uninfected macrophages.
This experiment was performed twice
with similar results.
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In situ hybridization
To examine the effects of TNF on the localized chemokine ex-
pression in the lungs early in infection, in situ hybridization was
performed to visualize the expression of CXCL9 and CXCL10. An
algorithm was designed to accurately assess the location and quan-
tity of gene expression with respect to pathology within the lung
(Table II). Single cells or nonclustered cells (20 cells together)
that expressed the gene of interested were classified as category 1.
Infiltrating clusters of cells were classified as category 2 or 3 on the
basis of histology and regardless of gene expression. Category 2
infiltrates were clusters or infiltrates of 20 100 cells, whereas cat-
egory 3 infiltrates were defined as clusters of 100 cells. At the
early time points after low dose aerosol infection (before day 14),
many of the infiltrates were near blood vessels, with little apparent
organization in both control and anti-TNF Ab-treated mice. After
classifying the infiltrates, the expression of genes was analyzed in
each infiltrate. If no signal was detected for the gene of interest the
infiltrate was designated negative. If signal was detected above
FIGURE 7. In TNF-deficient mice, chemokine expression was transiently reduced in CD11b cells isolated ex vivo from the lungs. A, Ex vivo isolation
of CD11b cells was performed on the lungs of TNFRp55/ () and WT (f) mice after high dose (300 CFU) aerosol M. tuberculosis infection. Real-time
RT-PCR was performed on the RNA from those cells on days 4, 7, and 10 (not shown) postinfection for expression of chemokines. B, Chronically infected
mice were treated with MP6XT-22 or IgG, and CD11b cells were harvested from lungs at various times post-treatment. Relative units are relative to
CD11b cells from uninfected lungs (A) or day 0 of Ab treatment (B; represented by line). CD11b cells from four or five mice per time point were pooled
to prepare RNA. This experiment was repeated, and similar results were obtained.
Table II. Algorithm used for ISH analysisa
Infiltrate ISH Signal Description
1 Individual cells or a group of 20 cells negative for gene expression (normal tissue)1 Individual cells or a group of 20 cells positive for gene expression
2 Clusters of 20 100 cells, negative for gene expression2 Clusters of 20 100 cells, 50% of cells positive for gene expression2 Clusters of 20 100 cells, 50% of cells positive for gene expression3 Clusters of 100 cells, negative for gene expression3 Clusters of 100 cells, 50% of cells positive for gene expression
3 Clusters of 100 cells, 50% of cells positive for gene expression
a Twenty 20 fields were counted for each mouse. There were three or four mice per experimental group. Each infiltrate wascounted and categorized based on size. In addition, all signals for CXCL9 and CXCL10 were counted and classified based on thecellular infiltrate expressing them. ISH, in situ hybridization.
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background, but 50% of the cells within a cellular aggregation
were positive for expression, the infiltrate was designated positive,
but if50% of the cells within a cellular cluster were signaling for
expression, the infiltrate was considered double positive. Therefore
each category 2 or 3 infiltrate could be either negative, positive, or
double positive.
The number of category 1 areas expressing CXCL9 or CXCL10
was counted in 20 20 fields. Category 1 (normal tissue) expres-
sion of CXCL10 was detected as early as 3 days postinfection, and
the expression increased gradually as infection progressed. By 12
days postinfection the number of category 1 areas expressing
CXCL10 (in the 20 fields counted) increased to an average of 8
2 in the IgG-treated mice and 4 1 in the anti-TNF Ab-treated
mice. Expression of CXCL9 was detected on day 6 in some, but
not all, mice. By 12 days postinfection the number of category 1
areas counted was 4 1 in the IgG-treated mice and 1 0.8 in the
FIGURE 8. The expressions of CXCL9 and CXCL10 are reduced in MP6XT-22-treated mice during acute aerosol infection. A, In situ hybridizations
were performed on fixed frozen sections of lung tissue. The expression of CXCL10 was reduced in infiltrates of intermediate size (category 2) in
MP6XT-22-treated mice. The expression of CXCL9 was reduced in larger category 3 in filtrates in MP6XT-22-treated mice compared with WT mice. B,
Each panel represents an example of the categorization used in the algorithm described in Table II.
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anti-TNF Ab-treated mice in the 20 fields counted. These data
suggest that TNF could affect early local CXCL9 and CXCL10
expression before granuloma formation.
The overall number of category 2 and 3 infiltrates was not dif-
ferent between MP6-treated and control mice (data not shown) up
to 12 days postinfection. This was not surprising, because at these
early time points the overall number of cells migrating into the
lungs was not significantly affected by anti-TNF Ab treatment
(Fig. 2), and this is before true granuloma formation. The number
of CXCL9 category 3 double-positive clusters was significantly
lower in the anti-TNF Ab-treated mice by 12 days postinfection,
but there were no differences in the number of CXCL9 category 2
positive clusters (Fig. 8 and data not shown). CXCL10 gene ex-
pression followed a different pattern; there were significantly fewer
CXCL10 category 2 positive clusters in MP6-treated mice, but no
differences in the number of CXCL10 category 3 positive clusters
(Fig. 8 and data not shown). These data confirm that TNF neu-
tralization affects the expression of a subset of chemokines in the
lung, and that effects on the timing of chemokine expression may
be important for granuloma formation.
DiscussionTNF is required for granuloma formation, and the absence of TNF
affects the expression of many inducible chemokines in the acute
and chronic models of tuberculosis. In the absence of TNF, CCR5
and CXCR3 ligand expression was reduced both in vivo and in
vitro, and the reduced expression was specific and localized to
macrophages and CD11b cells. In the absence of TNF, the cell-
mediated immune response fails to control infection, and the bac-
terial burden increases. Interestingly, in the chronic model of in-
fection, the mice succumb to infection before the bacterial burdens
rise to moribund levels, indicating that lung pathology is leading to
a decrease in lung function and death (12). Deciphering the dif-
ference between the influences of TNF and bacterial burden on
chemokine expression in a model in which TNF affects bacterial
burden was technically challenging. Focusing attention at earlytime points in the experimental models allowed us to diminish the
impact of higher bacterial burdens in TNF-deficient mice. In ad-
dition, we focused on macrophage chemokine expression because
this cell is a major M. tuberculosis reservoir and is responsive to
TNF. This enabled detection of the effects of anti-TNF Ab on
chemokine expression before changes in pathology, granuloma
formation, and increased bacterial burden. After increased bacte-
rial burdens, the chemokine gradients were even more dramatically
altered. The initial changes in chemokine expression may lead to
a cascade of inappropriate cellular communication, impaired cell
migration within the lung, loss of cell-mediated immune response,
and substantial lung pathology.
Control of cell migration is a complex process involving notonly chemokines and receptors, but also adhesion molecules. The
complexity of this process has been illustrated in this study. We
also examined the expression of adhesion molecules using gene
expression filter arrays (GEArray Q Series; SuperArray Bio-
science, Frederick, MD), but no striking differences were observed
after anti-TNF Ab treatment (data not shown). Clearly, changes in
pathology are compounded by the other roles that TNF plays in
control ofM. tuberculosis infection, and the effects on chemokine
expression are probably not solely responsible for the severe pa-
thology observed in TNF-deficient mice. Studies on low dose aero-
sol M. tuberculosis infection in CCR2 and CCR5 chemokine re-
ceptor transgenic knockout mice (Ref. 20 and data not shown) and
in vivo models of CXCL9 and CXCL10 neutralization (Ref. 28
and data not shown) have revealed some differences in cell migra-
tion, but even in these models the pathology does not mirror the
destructive pathology observed in the absence of TNF, where sig-
naling by some of the same chemokines that were affected by TNF
neutralization is diminished. CCR2/ mice have fewer macro-
phages throughout infection, but have sufficient numbers to control
low dose infection, although the mice are quite susceptible to high
dose infection (20, 29). CCR5/ mice had a greater lymphocytic
infiltrate in their lungs, but controlled infection.4 CXCL10/ and
mice treated with both anti-CXCL9 and anti-CXCL10 Abs con-
trolled infection with no apparent lymphocyte migration defi
cien-
cies (H. M. Scott Algood, J. L. Flynn, and A. D. Luster, unpub-
lished observations) to the lung, but a slight delay in lymphocyte
migration to the lymph nodes.
A recently published study provided data that anti-CXCL9 Ab
treatment in M. tuberculosis-infected mice did not affect the con-
trol of infection, but the mice had smaller, although still organized,
granulomas (28). These authors suggested that CXCL9 was
produced by neutrophils and played a key role in granuloma for-
mation. However, the differences we observed in pathology and
granuloma formation in TNF-deficient mice were not found in the
anti-CXCL9 Ab-treated mice. Therefore, TNF-mediated control of
CXCL9 expression may be only one factor in the influence this
cytokine has on granuloma formation.
Our studies on whole lung chemokine RNA expression consis-
tently (in three experiments) revealed differences at early time
points postinfection and after Ab treatment when comparing con-
trol and TNF-deficient mice, although the differences were not
statistically significant (data not shown). The LCM technique was
used to examine more localized gene expression patterns in gran-
ulomas. To isolate enough RNA from fixed, embedded tissue to
perform real-time RT-PCR on the LCM samples, multiple granu-
lomas had to be isolated from each mouse. Although the granulo-
mas isolated were from the same lobe of the lung, there was still
high variability among mice and probably among granulomas, be-
cause granuloma size and the number of bacteria in each granu-
loma can be variable (data not shown). Although the LCM data
revealed that chemokine expression was higher within the granu-loma compared with whole lung, the pattern of expression was
similar to whole lung chemokine expression.
We next focused our in vivo studies on the CD11b population
of cells, enriching for macrophages. With this technique we were
able to specifically localize reduced expression of CXCR3 and
CCR5 ligands to the CD11b cells, confirming our in vitro studies.
The technique used removed epithelial cells and lymphocytes from
the lung homogenates, but the neutrophils (which are CD11b)
were not removed. We have focused on the macrophages, because
direct effects of TNF on chemokine production by this population
was observed in vitro. However, TNF may also affect chemokine
production by neutrophils. The number of neutrophils at early time
points postinfection and after Ab treatment in chronically infectedmice was similar to the number of macrophages present in the
lungs(20) (Figs. 3 and 4). There were 35 104 macrophages and
35 104 neutrophils 6 days postinfection, and 4 105 of each
cell type at 4 days after initiation of Ab treatment in the chronic
model.
Recently, there has been published evidence of TNF affecting
chemokine levels in the liver during i.v. M. smegmatis and M.
tuberculosis infection (10, 11). Our study has gone beyond these
by focusing on localized chemokine expression in lungs after aero-
sol infection and by addressing chemokines that were not studied.
4 H. M. Scott Algood. Enhanced dendritic cell migration and T cell priming in the
lymph nodes result in increased lymphocytic infiltration in chemokine receptor 5(CCR5)-deficient mice following Mycobacterium tuberculosis infection. Submitted
for publication.
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We also carefully controlled for the confounding effects of bacte-
rial number on chemokine expression by studying very early time
points in the infection or neutralization. We demonstrated that che-
mokine deficiency in the absence of TNF was at the macrophage
level, using in vivo and in vitro studies. The macrophage probably
plays a key role in granuloma formation as an initial site of infec-
tion, a reservoir for M. tuberculosis, and a potent producer of both
TNF and chemokines.
The chemokines affected by TNF are known to infl
uence the
migration of cell types found within the lung during M. tubercu-
losis infection. CCL5, produced in the lung and by macrophages at
high levels, induces migration of T lymphocytes, monocytes, and
immature dendritic cells through CCR1 and CCR5. Disruption of
the CCL5 gradient, as in TNF-deficient mice, probably affects the
migration of these cells within the lung. T lymphocyte migration in
the lungs could also be affected by the reduction in CXCL9,
CXCL10, CCL3, and CCL4. Monocyte and macrophage migration
may be affected by similar chemokines, including CCL3, CCL4
through CCR5, CCL2, and CCL12 through CCR2. The increased
expression of these chemokines after increased bacterial load also
probably leads to increased cell migration to the lungs. Therefore,
we propose that initial reduction in chemokines in the absence of
TNF leads to localized changes in the gradients, decreased cell
responsiveness, and disorganized cellular infiltrate. As a result the
cells do not interact with each other in a coordinated manner, thus
reducing the cell-mediated response (11). The reduced cell-medi-
ated response leads to increased bacterial burden, increased che-
mokine expression, and an increase in cells migrating into the
lungs.
The chemokine expression studies performed in vitro advance
our understanding of the role of TNF in chemokine expression and
in post-transcriptional regulation of chemokines. Gene expression
of most chemokines tested diminished by 48 h postinfection (ex-
cept CCL5); this may indicate that there was a negative feedback
loop for gene expression. CCL3 mRNA expression peaked at a
similar point in anti-TNF Ab-treated and control macrophages, butmRNA levels remained higher in TNF-neutralized cultures. How-
ever, the protein level peaked at a lower point and was reduced in
TNF-neutralized cultures. Anti-TNF Ab-treated macrophages may
continue to express the gene in these cultures, because the protein
has not accumulated to a level to turn down transcription. In ad-
dition, the expression of CCL2 was only reduced very early after
infection in the presence of anti-TNF Ab, but protein expression
was reduced as late as 48 h postinfection. This suggests that CCL2
and CCL3 post-transcriptional regulation may be affected by TNF.
The in situ hybridization analysis of CXCL9 and CXCL10 ex-
pression highlighted the extensive variability between different
stages of cellular infiltrates, granuloma formation, and their che-
mokine expression. First, analyzing the number and size of theinfiltrates confirmed that at early time points cellular infiltration in
the lungs was not different in anti-TNF Ab-treated mice compared
with control mice. As infection progressed, control mice devel-
oped organized granulomas, whereas TNF-deficient mice did not.
Early patterns of chemokine expression presumably affect this or-
ganization of the cellular infiltrate. The size of the collection of
cellular infiltrate in the lungs can reasonably be attributed to the
time of formation; as the infiltrate grows and collects more cells,
the granuloma forms. The expression of CXCL10 in smaller
(early) infiltrates (category 2) was affected by TNF, but CXCL9
expression was only influenced by TNF in larger infiltrates (cate-
gory 3). This may suggest a differential timing of chemokine ex-
pression in granuloma formation, or that the expression of these
chemokines is sensitive to different levels of TNF. Alternatively, in
the larger infiltrates there are more cellular interactions and pos-
sibly more cytokines to signal CXCL10 expression, overcoming a
deficiency in TNF. In macrophages, CXCL9 was also more dra-
matically affected by anti-TNF Ab than was CXCL10. The kinetics
of chemokine expression are clearly complex in the lungs, and the
dynamics of granuloma formation may depend on TNF signaling
the expression of different chemokines at specific times.
In summary, this in-depth study of localized chemokine expres-
sion patterns during acute and chronic M. tuberculosis infections
presents evidence for TNF playing a direct role in chemokine ex-
pression and granuloma formation. We demonstrated that macro-
phages in vitro and CD11b cells in vivo are partially dependent
on TNF for the expression of many inducible chemokines after
M. tuberculosis infection. Our findings are relevant to understand-
ing the immune response and granuloma formation during aerosol
M. tuberculosis infection. Anti-TNF therapies have clearly illus-
trated the requirement for TNF in controlling latent tuberculosis in
humans, and our studies have defined one possible mechanism of
TNF in the lungs during infection. These studies will contribute to
an appreciation of the potential effects of anti-inflammatory or an-
tichemokine therapies on infections.
AcknowledgmentsWe are grateful to Amy Myers for excellent technical assistance, and to themembers of the Flynn, Chan, and Nau laboratories for helpful discussions.
We are grateful for the Reinhart laboratorys technical assistance with the
ISH protocol, particularly the guidance of Craig Fuller. We appreciate
Sergio Onate providing assistance and access to the laser capture micros-
copy equipment.
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