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MAJOR ARTICLE
Association between Interleukin-8 Gene Alleles
and Human Susceptibility to Tuberculosis Disease
Xin Ma,1 Robert A. Reich,1 John A. Wright,1 Heather R. Tooker,1 Larry D. Teeter,1 James M. Musser,3
and Edward A. Graviss1,2
Departments of 1Pathology and 2Medicine, Baylor College of Medicine, Houston, Texas; 3Laboratory of Human Bacterial Pathogenesis,
Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana
Interleukin (IL)–8 is involved in the pathogenesis of human tuberculosis (TB). However, the contribution of
polymorphisms of the IL-8 gene and its receptor genes CXCR-1 and CXCR-2 to human TB susceptibility
remains untested. In a case-control study, white subjects with TB disease were more likely to be homozygous
for the IL-8 ⫺251A allele, compared with control subjects (odds ratio [OR], 3.41; 95% confidence interval
[CI], 1.52–7.64). African Americans with TB also showed an increased odds of being homozygous for this
allele (OR, 3.46; 95% CI, 1.48–8.08). To exclude population artifacts in the case-control study, a separate
analysis that used a transmission-disequilibrium test with 76 informative families confirmed that the IL-8
⫺251A allele was preferentially transmitted to TB-infected children (P p .02 ). CXCR-1 and CXCR-2 did not
demonstrate significant associations with TB susceptibility. These data suggest that IL-8 is important in the
genetic control of human TB susceptibility.
Interleukin (IL)–8 is an important chemokine in the
human inflammatory process and functions as a potent
chemoattractant for the recruitment of leukocytes to
inflammatory sites [1–3]. In particular, the role of IL8 in human tuberculosis (TB) disease has become a
major focus for TB researchers worldwide. Initially,
clinical and pathological observations have shown remarkably elevated levels of IL-8 in tuberculous pleural
exudate [4], bronchoalveolar lavage fluid [5], and cerebrospinal fluid [6]. IL-8 also is expressed predominantly in tuberculous granulomas heavily infiltrated by
neutrophils [7]. In addition, several studies have demonstrated that IL-8 concentrations in plasma are higher
in patients who die from TB than in survivors [2, 8].
In response to anti-TB treatments, IL-8 in sputum
closely parallels and even precedes mycobacterial clearance in the sputum [9]. Further studies reveal that IL-
Received 8 January 2003; accepted 4 March 2003; electronically published 10
July 2003.
Financial support: National Institute of Allergy and Infectious Diseases, National
Institutes of Health (contracts N01-AO-02738 and AI-41168).
Reprints or correspondence: Dr. Edward A. Graviss, Dept. of Pathology (209E),
Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (egraviss@
bcm.tmc.edu).
The Journal of Infectious Diseases 2003; 188:349–55
2003 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2003/18803-0002$15.00
8 is produced and released by leukocytes [10–13] and
structure cells [13–16] in response to Mycobacterium
tuberculosis or its components. Pulmonary epithelial
cells, covering a 70-m2 surface area, are a major source
of IL-8 in the lungs [17]. Recent studies confirm that
expression of the IL-8 gene is up-regulated in human
macrophages infected with M. tuberculosis through the
protein tyrosine kinases and NF-kB [18] and comprises
part of the macrophage activation program [19]. In
vivo, anti–IL-8 antibody inhibits granuloma formation
in rabbits, which suggests that IL-8 may be central in
host defense to M. tuberculosis [20]. Two types of IL8 receptors, CXCR-1 and CXCR-2, have been identified and found to be present in equal ratios on all
polymorphonuclear leukocytes (PMNLs), monocytes
(CXCR 2 dominant), and 5%–25% of total lymphocytes (CXCR-2 dominant) [21]. Functional studies have
shown that both CXCR-1 and CXCR-2 mediate chemotaxis, the release of granule enzymes, and changes
in cytosolic free calcium ions. However, only CXCR-1
triggers activation of phospholipase D and the respiratory burst [22]. Strikingly, surface expression of
CXCR-1 and CXCR-2 is down-regulated on PMNLs
from human immunodeficiency virus (HIV) type 1–infected individuals and patients coinfected with M. tuberculosis and HIV-1 [23]. Down-regulation of both
IL-8 and Human Tuberculosis
• JID 2003:188 (1 August) • 349
receptors in PMNLs from infected individuals is directly related
to impaired IL-8–induced degranulation [24]. These findings
suggest that limited IL-8–dependent PMNL degranulation in
HIV-1–infected individuals may increase susceptibility to secondary microbial infections by organisms such as M. tuberculosis.
Recently, a population-based case-control study described an
association between systemic sclerosis and 2 polymorphisms
(CXCR-2 +785TrC, CXCR-2 +1208CrT) in the CXCR-2 gene
[25]. Family-based association studies suggested that a polymorphic allele (IL-8 ⫺251A) of IL-8, which shows a trend
toward an association with increased IL-8 production by lipopolysaccharide-stimulated whole blood, determined susceptibility and severity of bronchiolitis after respiratory syncytial
virus infections [26, 27]. At present, associations between these
polymorphisms in IL-8, CXCR-1, and CXCR-2 genes and human TB susceptibility remain untested. Within an ongoing population-based TB surveillance study, Houston Tuberculosis Initiative (HTI), we conducted a population-based case-control
study with adults with TB and control subjects without TB to
test the possible association between these polymorphisms in
IL-8, CXCR-1, and CXCR-2 genes and human TB susceptibility.
Subsequently, a family-based transmission-disequilibrium test
(TDT) with children with TB, and their parents and siblings
was performed to exclude bias and false association due to
population structure in the adult case-control study.
PATIENTS AND METHODS
Genetic polymorphisms in the IL-8, CXCR-1, and CXCR-2
genes and genotyping strategy. Nine sequence variants in the
IL-8, 1 in CXCR-1, and 3 in CXCR-2 gene regions have been
reported in the previous studies [25, 27]. The IL-8 polymorphisms are all located in the noncoding regions of the gene.
The ethnic difference on the allele frequencies of these polymorphisms has been observed [27]. The single-nucleotide polymorphism (SNP) in CXCR-1 is located in exon 2, resulting in
a conservative amino acid substitution from serine to threonine.
One SNP in exon 11 of CXCR-2 results in a synonymous codon
change, and the other 2 SNPs are located in noncoding regions
[25]. Some of these variants are either rarely present in populations, or in strong linkage disequilibrium [25]. On the basis
of implications from other studies [25–27] and our preliminary
data (not shown), we selected and analyzed only 1 SNP from
each of 3 genes (⫺251TrA in 5 promoter of IL-8, +2607GrC
in exon 2 of CXCR-1, and +785CrT in exon 11 of CXCR-2).
DNA was isolated from blood samples by use of the Nucleon
DNA extraction and purification kit (Amersham International
and Scotlab). Allele-specific PCR was used in genotyping of
subjects, as described elsewhere [25–27]. Results of allele-specific PCRs were analyzed independently in a blinded fashion
by 2 molecular biologists.
350 • JID 2003:188 (1 August) • Ma et al.
Adult case-control study. One hundred six adult white
patients with clinical TB disease (mean SD age, 51.2 11.2 years; 74.4% men) were collected from the HTI database,
including 92 patients with pulmonary TB, 11 with extrapulmonary TB, and 3 with both pulmonary and extrapulmonary
TB. All patients were identified by either M. tuberculosis culture
positivity (n p 103) or clinical improvement in response to
antimycobacterial treatment (n p 3 ). One hundred eighty adult
African American patients with TB (age, 46.6 9.7 years;
63.9% men) received a diagnosis via culture positivity (n p
174) and clinical improvement to anti-TB treatment (n p 6),
including 152 patients with pulmonary TB, 20 patients with
extrapulmonary TB, and 8 patients with both. A total of 107
whites (age, 56.4 9.8 years; 55.6% men) and 167 African
Americans (age, 41.9 10.9 years; 60.4% men) without a history of TB disease, autoimmune diseases, and other infectious
diseases were recruited from local hospitals and clinics as control subjects for this study. All patients and control subjects
were HIV seronegative. Race and ethnicity was determined for
each individual by self-identification. White was defined as a
non-Hispanic individual in this study. Frequencies of genotypes
between the case patients and control subjects in each ethnic
group were compared by x2 test for 2 ⫻ 2 contingency tables
with SAS software (version 8.0; SAS Institute). P values were
corrected for multiple comparisons by the formula Pcorr p
1 ⫺ (1 ⫺ P)n, where P is the uncorrected P value and n is the
number of comparisons. Pcorr ! .05 was considered to be statistically significant.
TDT within the families of children with TB. Findings
in the adult case-control study were verified by conducting a
family-based case-control study that used TDT [28]. One hundred thirty-one nuclear families, consisting of 159 children with
TB (table 1) and 469 family members, were enrolled and blood
samples drawn through the HTI. These cases were identified
Table 1. Demographic and clinical features of
children with tuberculosis (TB) from the Houston
Tuberculosis Initiative.
Characteristic
Value
Total no. children with TB
159
Age, mean years (range)
7.55 (0.17–18.00)
Male sex
75 (47.2)
Ethnicity
Hispanic
90 (56.6)
African American
47 (29.6)
Other race
22 (13.8)
Pulmonary TB
70 (44.0)
Lymphatic TB
56 (35.2)
TB at other sites
33 (20.8)
NOTE.
noted.
Data are no. (%) of subjects, except where
either by M. tuberculosis culture positivity (n p 76 ) or clinical
improvement while receiving TB treatment (n p 83 ) after contact investigation. Twenty families had 11 child with TB.
Twenty-one families did not included both parents but did
include at least 1 unaffected sibling, who could be analyzed by
the Sib-TDT (S-TDT) [29]. Because S-TDT is generally less
powerful than conventional TDT, we used conventional TDT
for those families that could be analyzed by either test. Finally,
we combined TDT and S-TDT into an overall test, as described
by Spielman et al. [29]. This study was approved by the Institutional Review Board for Baylor College of Medicine and
Affiliated Hospitals.
RESULTS
Association between the IL-8 5251A allele and adult clinical
TB disease. A total of 213 white and 347 African American
subjects from adult case and control groups were genotyped,
to identify specific SNPs: IL-8 ⫺251TrA, CXCR-1 +2607GrC,
and CXCR-2 +785TrC (table 2). The allele frequencies of these
SNPs showed notable ethnic divergence, a finding consistent
with previous reports [25, 26]. For example, the IL-8 ⫺251A
allele presents in whites with an allele frequency of 0.37, compared with 0.75 in African Americans. Nevertheless, all genotypic distributions from the different study groups conformed
to the Hardy-Weinberg equilibrium.
When comparing allele frequencies of these SNPs between
case and control groups, the IL-8 ⫺251A allele showed signif-
icantly higher frequencies in case groups than in control groups
(white, 0.52 vs. 0.37 [P ! .002]; African Americans, 0.79 vs.
0.71 [P ! .02]). No significant differences in allele frequencies
of CXCR-1 +2607GrC and CXCR-2 +785TrC were observed
between case patients and control subjects in either ethnicity.
To identify whether the IL-8 ⫺251A allele was associated with
adult clinical TB disease, we compared the distribution of IL8 ⫺251TrA genotypes between case patients and control subjects. In whites, homozygotes of the IL-8 ⫺251A allele presented
at a higher frequency in the case group than in the control
group (0.26 vs. 0.14; table 2) and showed an increased risk
(odds ratio [OR], 3.41; 95% confidence interval [CI], 1.52–7.64;
Pcorr ! .006), compared with homozygotes of the IL-8 ⫺251T
allele. Heterozygotes of IL-8 ⫺251A and T alleles also showed
a moderate risk (OR, 2.01; 95% CI, 1.06–3.80; Pcorr p .07),
compared with homozygotes of the IL-8 ⫺251T allele. Similar
risks for these genotypes were found among African Americans
(OR, 3.46 for homozygotes and 3.39 for heterozygotes), even
though frequencies of these genotypes were different between
the 2 ethnicities (table 2).
Linkage between the IL-8 locus and clinical TB disease in
children. TDT is a powerful test for linkage between a genetic
marker and a disease-susceptibility locus in the presence of an
association. Hence, to confirm the involvement of IL-8 in human TB susceptibility, we conducted a TDT analysis with the
131 nuclear families, each including at least 1 child with TB.
Of these families, 64 families with both parents available were
informative for conventional TDT analysis by having at least
Table 2. Associations between single-nucleotide polymorphisms (SNPs) in IL-8, CXCR-1, and CXCR-2 genes
and clinical tuberculosis (TB) in adults.
African American subjects
White subjects
SNP, genotype
IL-8 ⫺251TrA
TT
TA
Patients
with TB
(n p 106)
Control
subjects
(n p 167
Patients
with TB
(n p 180)
OR (95% CI)
42 (39.3)
50 (46.7)
23 (21.7)
55 (51.9)
1.0
2.01 (1.06–3.80)b
23 (13.8)
8 (4.4)
50 (29.9)
59 (32.8)
3.39 (1.40–8.25)c
15 (14.0)
28 (26.4)
3.41 (1.52–7.64)
c
94 (56.3)
113 (62.8)
3.46 (1.48–8.08)c
89 (83.2)
17 (15.9)
91 (85.8)
13 (12.3)
1.0
0.75 (0.34–1.63)
107 (64.1)
53 (31.7)
112 (62.2)
61 (33.9)
1.0
1.10 (0.70–1.73)
1 (0.01)
1 (0.01)
0.98 (0.60–15.88)
7 (4.2)
7 (3.9)
0.96 (0.32–2.82)
21 (19.6)
54 (50.5)
32 (29.9)
24 (22.6)
45 (42.5)
37 (34.9)
1.0
0.72 (0.36–1.48)
1.01 (0.48–2.15)
77 (46.1)
82 (49.1)
8 (4.8)
80 (44.4)
86 (47.8)
14 (7.8)
1.0
1.00 (0.65–1.56)
1.68 (0.67–4.24)
OR (95% CI)
a
AA
CXCR-1 +2607GrC
GG
GC
CC
CXCR-2 +785TrCa
TT
TC
CC
Control
subjects
(n p 107)
1.0
a
NOTE. Data are no. (%) of subjects. P values were corrected for multiple comparisons by the formula (Pcorr p 1 ⫺ (1 ⫺ P )n , where
P is the uncorrected P value and n is the number of comparisons). Pcorr ! .05 was considered to be statistically significant.
a
⫺251TrA, corresponding to the position 251 nt upstream of transcription start point of IL-8; +2607GrC, position 6334 of GenBank
sequence L19592; +785TrC, position 10657 of GenBank sequence M99412.
b
Pcorr p .07.
c
Pcorr ! .01.
IL-8 and Human Tuberculosis • JID 2003:188 (1 August) • 351
Table 3. Transmission of the IL-8 ⫺251A allele within 64 informative
families for conventional transmission-disequilibrium test.
Transmission
of IL-8 ⫺251A allele
Transmitted
Nontransmitted
Percentage
transmitted
Children with TB (np 72)
50
32
61
Siblings without TB (n p 74)
38
46
45
Affected status
NOTE.
a
b
TB, tuberculosis.
a
95% confidence interval, 51–71 (x2 p 3.95 ; P ! .05 ). These results were calculated by
conventional transmission-disequilibrium test analysis described elsewhere [29, 30].
b
The transmission of the IL-8 ⫺251A allele to siblings without TB was significantly
less frequent than to the siblings with TB (P p .04).
1 parent who was heterozygous for the IL-8 ⫺251TrA polymorphism. Specifically, in 53 families, 1 parent was heterozygous, and in 11 families, both parents were heterozygous. Seven
families had 11 affected child (multiplex family). A total of 82
transmissions of IL-8 ⫺251A or T alleles occurred from heterozygous parents to 72 children with TB, in which the IL-8
⫺251A allele was transmitted significantly more often than
expected (transmission percentage, 61%; 95% CI, 51%–71%;
P ! .05; table 3), whereas the proportion of the IL-8 ⫺251A
allele transmitted from heterozygous parents to unaffected children was 45%. The difference was statistically significant
(P p .04). Consequently, this result provided evidence for a
linkage between the marker IL-8 ⫺251TrA and TB susceptibility without evidence for segregation distortion.
In addition, 12 informative families with only 1 parent but
at least 1 unaffected sibling having a different genotype were
eligible for S-TDT analysis. The IL-8 ⫺251A allele was observed
more frequently among children with TB than among those
without TB (0.69 vs. 0.55) (table 4). Because of the small sample
size of only 12 sibships, we combined conventional TDT and
S-TDT as an overall test, as described by Spielman et al. [29].
The Z statistic was 2.04, inferring a P value of .02 by use of
the normal distribution approximation [29]. Therefore, this
result supports our finding in the adult case-control study and
implies that the IL-8 locus harbors a TB-susceptibility gene or
genes, most likely IL-8 itself.
DISCUSSION
Accumulated evidence indicates that IL-8 is an important mediator of host response to and pathogenesis of a variety of
microbes causing common human diseases, including TB.
However, a recent genomewide linkage study indicated no evidence for linkage between human TB susceptibility and loci
of IL-8 or its receptor genes CXCR-1 and CXCR-2 in African
blacks [31]. Nonetheless, the candidate gene–based association
study has been considered more powerful than the linkage study
in determination of genetic susceptibility to human complex
diseases such as TB disease [32]. In the present study, we used
352 • JID 2003:188 (1 August) • Ma et al.
2 strategies, a population-based association study (i.e., the casecontrol study) and a family-based association study (i.e., the
TDT) to explore contribution of IL-8, CXCR-1, and CXCR-2
genes in human TB susceptibility. The case-control study is
capable of detecting subtle genetic risks in complex diseases by
providing an estimate of relative risk (i.e., OR), but bias may
exist as a result of population admixture or stratification, existing in ethnically and racially mixed American populations.
In contrast, TDT that used within-family control subjects minimizes or eliminates this population structure bias but has the
limitation of being unable to measure relative risk directly. In
practice, as a test of linkage, TDT has shown robust power for
identification of markers closely linked to disease-susceptible
genes [28]. Therefore, the combination of these 2 complementary approaches has satisfied some criteria for reliable results
in genetic association studies in common diseases [33].
Our results from the adult case-control study initially suggested that the IL-8 ⫺251A allele was associated with an increased TB disease risk for its carrier with a dominant mode.
Moreover, an OR 13.0 for homozygotes of the IL-8 ⫺251A
allele, compared with homozygotes of the IL-8 ⫺251T allele,
was consistently observed in the 2 ethnicities. This finding suggests that IL-8 may play a more prominent role in TB susceptibility, compared with other TB-associated genes, such as
NRAMP1, which have polymorphisms with ORs !3.0 in our
studied population [34] and other populations [35, 36].
Second, the TDT result from 76 informative families suggests
that a linkage exists between the IL-8 locus and human susceptibility to TB disease by showing preferential transmission
of the IL-8 ⫺251A allele to the affected children. Taken together,
Table 4. Allele frequencies of IL-8 ⫺251A in 12 informative families for sibling transmission-disequilibrium test.
IL-8 ⫺251TrA
Affected status
⫺251A allele
⫺251T allele
Children with TB (n p 13)
18 (0.69)
8 (0.31)
Siblings without TB (n p 26)
31 (0.55)
21 (0.45)
NOTE.
Data are no. (% frequency) of alleles. TB, tuberculosis.
our data indicate linkage and association between IL-8 and
susceptibility to TB disease in adults and children. Thus, we
propose that IL-8 is one of the important genes governing
human genetic susceptibility to TB disease. In addition, higher
IL-8 ⫺251A allele frequency in African American than in white
subjects may contribute to a reported enhanced susceptibility
of African Americans to TB disease than whites when exposed
to similar environments [37]. It is notable that the IL-8 ⫺251A
allele presents different allele frequencies in our studied populations from the previous reports in the subpopulations of
Europe and Africa [26, 27]. These variations are likely the result
of the heterogeneity of gene pool of ethnically and racially
mixed American populations. Of interest, our family-based
TDT analysis systematically rule out the possibility of biased
associations in the population-based case-control study. To help
minimize possible self-selection bias in recruiting control subjects
from hospitals and clinics, we used the control recruitment criteria of no history of TB disease, autoimmune diseases, or other
infectious diseases. In addition, the similar relative risks of IL-8
⫺251A genotypes between white and African American subjects
also decreased the likelihood of an artificial association between
IL-8 gene and human genetic susceptibility to TB disease.
Functional studies on the 5 flanking region of the IL-8 gene
reveal several binding sites for transcriptional factors activator
protein–1, NF–IL-6, and NF-kB, which are sufficient for maximal transcriptional responses to most proinflammatory mediators [38]. Of interest, all these binding sites are located
within a narrow range of 100 nt and are close to the transcription start point (⫺130 nt) of IL-8. Only 2 rare SNPs in this
region of IL-8 are registered in the GenBank database, dbSNP,
indicating conservation and functional importance of the promoter. However, IL-8 ⫺251TrA (⫺251 nt upstream of the
transcription start point) is located outside of this region, and
it is in an area not characterized as a transcription factor binding site.
Recently, a TDT study suggested that an unusual haplotype
of IL-8 ⫺251A/781T (781T/C, another SNP in intron 1 of IL8), rather than IL-8 ⫺251A itself, was linked with genetic predisposition to respiratory syncytial virus infection [27]. However, our data from patients with TB showed that this haplotype
was not preferentially transmitted to the affected children (52%
transmitted; 95% CI, 46%–58%; P p .60). A further study with
a larger sample size is necessary to clarify the association between this particular haplotype and human TB susceptibility.
To date, only one in vitro study suggests that IL-8 ⫺251TrA
alters IL-8 expression (i.e., expression was increased) [26].
Why might a higher expression of IL-8 cause a risk to clinical
TB? One explanation may be that increased expression of IL8 attracts an excess of leukocytes to the disease site, resulting
in extensive tissue damage, often seen in pulmonary TB by
generation of damaging free radicals, proteases, and elastases.
Recent observations confirmed that the high IL-8 secretion
could also enhance the inflammation by delaying the apoptosis
of PMNLs [39, 40]. At this point, the outcome of M. tuberculosis
infection may not only depend on the virulence of M. tuberculosis, but also the host inflammatory response. Therefore, the
IL-8 ⫺251A allele or other functional polymorphisms in strong
linkage disequilibrium with the IL-8 ⫺251A allele are in need
of further investigation—in particular, because 10 of the human
CXC chemokine genes are also physically mapped on the same
chromosome region 4q in addition to IL-8 [41].
Neither genotypes nor allele frequencies of CXCR-1
+2607GrC and CXCR-2 +785TrC showed significant difference between case patients with TB and control subjects without
TB. CXCR-1 +2607GrC results in a conservative amino acid
substitution from serine to threonine at the 276 amino acid
residue of CXCR-1, which is located between the sixth and
seventh putative transmembrane domains [42]. It is unlikely
that this substitution makes a significant change in the structure
and function of the CXCR-1 protein. CXCR-2 +785TrC is a
silent substitution in exon 11 of the CXCR-2 gene. An association between systemic sclerosis and CXCR-2 +785TrC was
detected in a case-control study, indicating it might be in strong
linkage disequilibrium with other functional polymorphisms
[25]. In addition to IL-8, CXCR-2 can bind other chemokines
with high affinity, such as the growth-related oncogene proteins
[43] and neutrophil-activating peptide 2 [44]; CXCR-1 does
not. Moreover, CXCR-2 is predominantly expressed on monocytes and IL-8 receptor-positive lymphocytes [21]. These observations imply that CXCR-2 mediates functions of IL-8 different from CXCR-1 and may be involved in the pathogenesis
of certain diseases such as systemic sclerosis, rather than other
diseases such as TB. The lack of association with the CXCR
receptor SNPs could also be the result of the small sample size;
thus, additional studies with larger population pools are needed
to confirm these findings.
In summary, our results indicate an association between
IL-8 and human genetic susceptibility to TB. The design of our
study satisfied the criteria of a reliable genetic association study.
For instance, results in the case-control study were independently obtained and confirmed from 2 ethnicities. Subsequently, the finding from the population-based study was supported by results from the family-based association study.
Hence, the potential biases from either of both methods have
been minimized.
Acknowledgments
We thank Kimmo Virtaneva and Frank DeLeo for their critical review of a draft of the manuscript.
IL-8 and Human Tuberculosis • JID 2003:188 (1 August) • 353
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