Gonorrhea remains highly prevalent worldwide. In the United States, gonorrhea is the second most commonly reported bacterial sexually transmitted infection, with more than 330,000 cases reported in 2004, and nearly as many new infections not reported annualy.1,2 Antibiotics recommended by the Centers for Disease Control and Prevention3 usually eradicate gonorrhea, but reinfection is common, suggesting that gonococcal infection in humans may fail to elicit long-lasting protective immunity, at least in some patients with modest levels of serum and genital mucosal antigonococcal antibodies after infection.4–10
Studies on cellular immune responses in gonorrhea are sparse. In vitro and experimental studies of gonorrhea in humans (i.e., intraurethral challenge with Neisseria gonorrhoeae) have generally demonstrated that proinflammatory cytokines predominate during acute infection.11–13 It remains unclear if such responses are applicable to genital mucosa in patients with sexually acquired gonorrhea.14,15
Genetic factors, including human leukocyte antigen (HLA) alleles and/or cytokine gene polymorphisms, may account for variable immune responses to gonorrhea. HLA alleles contribute to the specificity of T-cell responses to gonococcal antigens, whereas cytokine gene polymorphisms can regulate cytokine production following immune activation. Among the sparse studies examining the relationship of genetic determinants to gonorrhea, Ness et al.16 evaluated data from the PEACH study of US women with clinically evident pelvic inflammatory disease. In a subset of patients, they found a borderline positive association of DQA1*0,301 and negative association of DQA1*0,102 with gonococcal cervicitis.16 Conceivably, these HLA class II alleles may differ in their ability to induce immune responses, especially humoral, to gonorrhea.
We previously investigated immunogenetic correlates of chlamydia in a cohort of adolescents enrolled in the Reaching for Excellence in Adolescent Care and Health (REACH) study.17,18 We subsequently assessed immunogenetic correlates of gonorrhea from this cohort and summarize our findings here.
Materials and Methods
Study Population and Data Collection
The REACH cohort comprised HIV-infected and high-risk HIV-uninfected male and female adolescents (13–18 years old) who were enrolled at 15 locations in 13 US cities.17,19–21 Longitudinal data were collected quarterly from 1996 to 2000, and patients with sufficient (6–56 months) follow-up, specimens for genetic analyses, and other relevant data were analyzed. Cytokine data were only available for female subjects.
N. gonorrhoeae Detection and Treatment
At enrollment and every 6 months thereafter, subjects were screened for N. gonorrhoeae (and Chlamydia trachomatis) by ligase chain reaction (LCx STD system, Abbott Laboratories, Abbott Park, IL).17,19 Test results from a central laboratory were returned to clinicians at variable time points (up to a 6-month interval). Treatment could be initiated before centrally obtained results became available because local testing was performed. Infected subjects were prescribed appropriate therapy.
DNA Extraction and HLA Typing
DNA extraction and HLA genotyping methods have been reported.17,22,23 Briefly, DNA was extracted from peripheral blood mononuclear cells for polymerase chain reaction (PCR)-based HLA typing: HLA-A, -B, and -C variants to 2-digit (group) specificity and DRB1 and DQB1 to 4-digit (allele) specificity.17,22,23 Haplotypes were assigned manually based on well-documented patterns of linkage disequilibria seen in various North American populations.24–27
Classification of TH1 and TH2 Cytokine Gene Polymorphisms
Sixteen common single nucleotide polymorphisms (Supplemental Table) from 7 genes (IL2, IL4, IL4R, IL6, IL10, IL12B, and TNF) representing TH1 and TH2 pathways were typed by PCR with sequence-specific primers.21 Validation of techniques and individual genotyping followed procedures recommended by the manufacturer (Department of Transplantation Immunology, University of Heidelberg, Heidelberg, Germany and Pel-Freez Clinical Systems, LLC, Brown Deer, WI) and the 13th (2002) International Histocompatibility Workshop.21,28,29 When multiple single nucleotide polymorphisms within a single gene were tested, cis and trans relationships were established by PCR for all except 2 at the IL6 locus. The insertion or deletion variants in IL12B promoter sequence corresponded to 2 alleles (longer, L and shorter, S) defined by fragment sizes that differ by 4-bp after PCR amplification and automated denaturing gel electrophoresis on the ALFexpress system (Amersham Pharmacia Biotech, Piscataway, NJ).29,30
Endocervical Cytokine Quantification
Endocervical secretions were collected every 6 months in a Weck-cel sponge placed in the cervix following speculum insertion as reported,31 and sponges were stored at −70°C. Before extracting secretions, sponges were weighed and equilibrated in phosphate buffered saline (PBS) + 0.25 mol/L NaCl with 10% Aprotinin (Sigma) for 30 minutes at 4°C. Secretions were extracted from each sponge by centrifuging at 12,000g for 20 minutes using a spin-x centrifuge filter unit. A previously described dilution factor for the final extract32 was used to calculate the final cytokine concentration when quantitative enzyme-linked immunosorbent assay kits (BioSource International, Camarillo, CA) were used to measure IL-2, IL-10, and IL-12 concentrations33; the lower detection limits were 5, 0.2, and 0.8 pg/mL, respectively.34 Other cytokines were not measured in REACH. We previously reported endocervical IL-2, IL-10, and IL-12 levels in chlamydia.18,35 In the analyses reported here, we focused on the relationship of these cytokines to gonorrhea, comparing cytokine levels at visits with gonorrhea and chlamydia coinfection, with either infection alone, or with neither present.
We used SAS (Statistical Analysis Software, Version 8.5; SAS Institute, Cary, NC) for analyses. Individuals were classified as having had gonorrhea if they tested positive by gonococcal ligase chain reaction at least once. The relationships of gonorrhea to nongenetic (sociodemographic and clinical) factors (collected at baseline) and genetic markers were assessed univariately (χ2 or Fisher exact tests) and with forward stepwise multivariable logistic regression.
Analyses of gonorrhea and other infection categories with endocervical cytokine responses were initially performed by selecting a single visit for each subject whose infection could be categorized as defined above (gonorrhea and chlamydia coinfection, gonorrhea or chlamydia alone, or neither). In subjects with visits in more than 1 of these categories, assignment was made to a single category in the priority order listed above based on the category frequency (i.e., preference was given to coinfection as the least frequent category, then gonorrhea alone, etc.). For subjects with more than 1 visit in a chosen category (e.g., multiple visits with gonorrhea alone), a single visit was selected for study randomly (by assigning a random number to each visit and selecting the visit with the lowest random number); we performed additional analyses to verify the randomization by choosing a different visit from the multiple same infection category visits and found it to be effective with no difference in study findings (these verification analyses are not shown). Because cytokine levels did not follow a normal distribution,36 median cytokine levels were assessed by the Kruskal-Wallis test. We subsequently tested log10-transformed IL-2, IL-10, and IL-12 levels in multivariable repeated measures (multiple-visit) regression models, in which distinctive infection categories and genetic and nongenetic factors were evaluated for independent effects.
Characteristics of Study Participants and Relationship to N. gonorrhoeae Infection
Clinical and genetic data were completed for 485 adolescents with characteristics previously reported.17 Subjects were predominantly female (74%), black (70%), and HIV-positive (68%), with a mean age of 17 years and median follow-up of 35 (range 6–56) months. An average of 6 (±2) N. gonorrhoeae and C. trachomatis tests were performed, and were positive at least once in 104 (21%) and 199 (41%) of subjects, respectively.
Subjects with versus without gonorrhea differed in relation to sociodemographic and clinical characteristics (Table 1). Those with gonorrhea were more likely to be black (P = 0.015), ≥17 years or older (P =0.0002), HIV-positive (P = 0.001) with CD4+ T-cell counts ≥400/μL, and on highly active antiretroviral therapy; they also more frequently acquired chlamydia (P <0.0001) and had more gonococcal tests performed and longer follow-up (P = 0.002 and P = 0.0008, respectively) (duration of follow-up was retained as a covariate in subsequent analyses as it could be universally applied to all subjects).
Genetic Variants Associated with N. gonorrhoeae Infection—Univariate Analyses
The −1902G variant in interleukin-4 receptor gene (IL4R) and the interleukin-2 gene (IL2) haplotype defined by −330T and 166G (IL2 T-G) were present more often in those with gonorrhea, whereas the −330G and −166T variants in IL2 as well as the IL2 T-T and G-G haplotypes were present less often (all P <0.05) (Table 2). Other cytokine gene variants or haplotypes present in ≥5% of patients were not associated with gonorrhea status. In addition, 4 HLA alleles (A*23, B*53, Cw*04, and DRB1*15) and 3 haplotypes (B*53-Cw*04, B*44-Cw*04, and DRB1*15-DQB1*06) were present more often in subjects with gonorrhea (Table 2), whereas 2 alleles (A*02 and DQB1*05) were present less often (all P <0.05) (Table 2). DQB1*06 showed a borderline association with gonorrhea (P = 0.052). The stronger association of gonorrhea with DRB1*15 versus DQB1*06 suggested that the DRB1*15-DQB1*06 haplotypic effect was mostly driven by DRB1*15, although strong linkage disequilibrium precluded a clear separation of allelic effects. Other HLA determinants present in ≥5% of the population were not associated with gonorrhea status.
Relationship of Genetic and Nongenetic Determinants to N. gonorrhoeae Infection as Defined in Multivariable Models
Individual genetic and nongenetic factors associated with gonorrhea in univariate analyses were tested in a multivariable logistic regression model (Table 3). Neither race nor several genetic determinants remained significantly associated with gonorrhea when examined jointly. In the final (reduced) model, gonorrhea was significantly more likely in subjects age ≥17 (P = 0.0002), HIV-1 infected (P = 0.011), with longer follow-up (P = 0.007), with chlamydia during the study (P <0.0001), with the IL2 T-G haplotype (P = 0.012), and with Cw*04 (P = 0.008). Subjects with DQB1*05 were significantly less likely to have gonorrhea (P = 0.022).
Relationship of Endocervical Cytokine Concentrations to N. gonorrhoeae Infection and Comparison With Those with C. trachomatis Infection, N. gonorrhoeae and C. trachomatis Coinfection, and No infection
In analyses of females with complete cytokine data (n = 388; the analyses also included some females not included in the above genetic analyses because of incomplete genetic data), we evaluated endocervical IL-2, IL-10, and IL-12 concentrations at a single visit for each female in 1 of the 4 infection categories: gonorrhea and chlamydia coinfection, gonorrhea or chlamydia alone, or neither (see the Materials and Methods section). For each cytokine, the median concentration differed significantly for each different infection category (Table 4). IL-2 levels were highest in those without chlamydia or gonorrhea and lowest in those with coinfection. IL-10 levels were lowest in those with neither infection and highest in those with coinfection. IL-12 levels were lowest in those with neither infection but similar in those with single- and coinfection. A subanalysis of 48 females with gonorrhea alone who then cleared infection within 2 consecutive visits (a visit with gonorrhea followed by a subsequent visit without gonorrhea detected) revealed that median IL-2 levels increased (217.5 vs. 234.6 pg/mL) while median IL-10 and IL-12 levels decreased (95.9 vs. 43.5 pg/mL and 340.7 vs. 278.5 pg/mL, respectively) after gonorrhea clearance; only the IL-10 decrease reached statistical significance (P = 0.005).
We performed multivariable analyses on all females using repeated measures with log10-transformed endocervical IL-2, IL-10, and IL-12 concentrations as dependent variables and independent covariates that included the 4 infection categories, relevant genetic determinants, and other nongenetic factors that could influence cytokine levels [including other STIs, demographic and behavioral factors, oral contraceptive use, and immune status (HIV infection and CD4+ T-cell count)]. After controlling for covariates (Table 5): 1) IL-2 concentrations were significantly lower (P = 0.004) in those with chlamydia alone (P = 0.002) or coinfection (P = 0.004) compared with no chlamydia or gonorrhea; IL-2 levels were also somewhat lower in those with gonorrhea alone compared with neither infection (P = 0.09); 2) IL-10 levels were significantly higher in those with gonorrhea and/or chlamydia than in those with neither (all P ≤0.01); 3) IL-12 levels were significantly higher in those with gonorrhea and chlamydia coinfection versus neither infection (P = 0.01); 4) HLA Cw*04 and DQB1*05 were not significantly associated with cytokine levels, except for a trend toward higher IL-2 concentrations in those with DQB1*05 (P = 0.055); and 5) the IL2 T-G haplotype was significantly associated with lower IL-2 levels (P = 0.012). The magnitude of the cytokine responses was similar in gonorrhea alone versus chlamydia alone, with differences in P values mainly because of sample size.
Further, in a subset of subjects (n = 43) who acquired gonorrhea within 2 consecutive visits (a visit with no gonorrhea followed by a subsequent visit with gonorrhea detected), those with the IL2 T-G haplotype (n = 39) had lower median IL-2 concentrations after gonorrhea acquisition (246.9 vs. 293.6 pg/mL), whereas those without IL2 T-G (n = 4) had higher median IL-2 concentrations after gonorrhea acquisition (161.6 vs. 132.7 pg/mL); these differences were not statistically significant (P = 0.29 for analyses of log10 IL-2 concentrations).
Our analyses identified associations of several genetic variants with gonorrhea in males and females and patterns of mucosal cytokine responses to gonorrhea in females. A single interleukin-2 gene (IL2) haplotype (T-G) and 2 HLA variants, Cw*04 and DQB1*05, were independently associated with gonorrhea. A possible link between mucosal IL-2 levels in females and a haplotype in the gene encoding IL-2 is of particular interest. Unfortunately, cytokine data were not available for males.
Comparison of endocervical cytokine levels in females with and without gonorrhea produced clear evidence that IL-2, a T-cell growth factor and a pro-TH1 cytokine, may be a limiting factor in immune responses to N. gonorrhoeae. The IL2 T-G haplotype, associated with a threefold higher risk for gonorrhea in our study, has previously been associated with reduced production of IL-2,37 and the latter relationship was also demonstrated in our repeated measures analyses (Table 5). In contrast, a slightly increased IL-2 response was seen in females with DQB1*05, which was associated with a twofold lower risk for gonorrhea. Such genetically mediated differences in mucosal IL-2 responses following N. gonorrhoeae infection are consistent with the important role IL-2 can play in regulating antigen-specific T-cells, B-cells, and NK cells.38,39 Overall, it seemed that a blunted IL-2 response related to the IL2 T-G haplotype or enhanced IL-2 response partially attributable to DQB1*05 may impair or boost expression of TH1 cytokines and the ability of NK cells to effectively prevent N. gonorrhoeae infection. It is also possible that other nongenetic factors, including other cytokines, may influence IL-2 levels.
Endocervical IL-10 and IL-12 concentrations were not associated with specific cytokine or HLA genotypes in our study. Of note, in females coinfected with chlamydia and gonorrhea, IL-2 and IL-10 levels showed additive effects, with a greater decrease and increase in respective levels of IL-2 and IL-10 when compared with others with either infection alone. These mucosal pathogens, which both infect genital tract columnar epithelial cells, seem to elicit similar immune responses in females, despite clear differences in genetic correlates. Thus, different mechanisms can drive and induce similar immunologic phenotypes.
The only previously published study (the PEACH cohort) that evaluated influences of HLA on gonorrhea16 differed from ours in that it targeted adolescent and adult females with clinical evidence of pelvic inflammatory disease, in contrast to the HIV-infected and high-risk HIV-uninfected adolescents with uncomplicated gonorrhea in our study. From the PEACH cohort, DQA1*0,102 and DQA1*0,301 were associated with gonococcal cervicitis16; these DQA1 alleles are known to be in strong linkage disequilibrium with DQB1*05/*06 and DQB1*03, respectively.24 As DQB1*05 was implicated in our study, the haplotype including DQA1*0,102 and DQB1*05 may be a favorable factor in both adolescent and adult populations. Further, the association of HLA-Cw*04 with increased risk of gonorrhea we identified has not been reported elsewhere. This novel relationship is worth further investigation as Cw*04 is a common variant in all major human populations.
Certain genetic relationships seen here were less likely than others to have been observed by chance in view of previous studies by others (e.g., DQA1 in gonorrhea16) and by us (e.g., IL-2 in chlamydia35). Clearly, those relationships that appear consistent with reported evidence deserve greater attention than those without (e.g., Cw*04). With overrepresentation of HIV-positive patients in our population, the immunogenetic factors identified in this study could be more relevant to studies involving immunosuppressed patients. It will be important to repeat such studies in HIV-negative immunocompetent patient cohorts.
1. Centers for Disease Control and Prevention. Sexually Transmitted Disease Surveillance, 2004. Atlanta, GA: U.S. Department of Health and Human Services, September 2005.
2. Weinstock H, Berman S, Cates W Jr. Sexually transmitted diseases among American youth: Incidence and prevalence estimates, 2000. Perspect Sex Reprod Health 2004; 36:6–10.
3. Centers for Disease Control and Prevention. Sex Transm Dis Guidelines, 2006. MMWR 2006; 55:42–49.
4. Plummer FA, Chubb H, Simonsen JN, et al. Antibody to Rmp (outer membrane protein 3) increases susceptibility to gonococcal infection. J Clin Invest 1993; 91:339–343.
5. Plummer FA, Chubb H, Simonsen JN, et al. Antibodies to opacity proteins (Opa) correlate with a reduced risk of gonococcal salpingitis. J Clin Invest 1994; 93:1748–1755.
6. Hedges SR, Mayo MS, Mestecky J, et al. Limited local and systemic antibody responses to Neisseria gonorrhoeae
during uncomplicated genital infections. Infect Immun 1999; 67:3937–3946.
7. Brooks GF, Lammel CJ. Humoral immune response to gonococcal infections. Clin Microbiol Rev 1989; 2:S5–10.
8. O'Reilly RJ, Lee L, Welch BG. Secretory IgA antibody responses to Neisseria gonorrhoeae
in the genital secretions of infected females. J Infect Dis 1976; 133:113–125.
9. McMillan A, McNeillage G, Young H, et al. Secretory antibody response of the cervix to infection with Neisseria gonorrhoeae.
Br J Vener Dis 1979; 55:265–270.
10. McMillan A, McNeillage G, Young H. Antibodies to Neisseria gonorrhoeae
: A study of the urethral exudates of 232 men. J Infect Dis 1979; 140:89–95.
11. Naumann M, Wessler S, Bartsch C, et al. Neisseria gonorrhoeae
epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines. J Exp Med 1997; 196:247–258.
12. Ramsey KH, Schneider H, Cross AS, et al. Inflammatory cytokines produced in response to experimental human gonorrhea. J Infect Dis 1995; 172:186–191.
13. Harvey HA, Post DM, Apicella MA. Immortalization of human urethral epithelial cells: A model for the study of the pathogenesis of and the inflammatory cytokine response to Neisseria gonorrhoeae
infection. Infect Immun 2002; 70:5808–5815.
14. Anzala AO, Simonsen JN, Kimani J, et al. Acute sexually transmitted infections increase human immunodeficiency virus type 1 plasma viremia, increase plasma type 2 cytokines, and decrease CD4 cell counts. J Infect Dis 2000; 182:459–466.
15. Hedges SR, Sibley DA, Mayo MS, et al. Cytokine and antibody responses in women infected with Neisseria gonorrhoeae
: Effects of concomitant infections. J Infect Dis 1998; 178:742–751.
16. Ness RB, Brunham RC, Shen C, et al. PID Evaluation Clinical Health (PEACH) Study Investigators. Associations among human leukocyte antigen (HLA) class II DQ variants, bacterial sexually transmitted diseases, endometritis, and fertility among women with clinical pelvic inflammatory disease. Sex Transm Dis 2004; 31:301–304.
17. Geisler WM, Tang J, Wang C, et al. Epidemiological and genetic correlates of incident Chlamydia trachomatis
infection in North American adolescents. J Infect Dis 2004; 190:1723–1729.
18. Wang C, Tang J, Geisler WM, et al. Human leukocyte antigen and cytokine gene variants as predictors of recurrent Chlamydia trachomatis
infections in high-risk adolescents. J Infect Dis 2005; 191:1084–1092.
19. Wilson CM, Houser J, Partlow C, et al. The REACH (Reaching for Excellence in Adolescent Care and Health) project: Study design, methods, and population profile. J Adolesc Health 2001; 29:8–18.
20. Tang J, Wilson CM, Meleth S, et al. Host genetic profiles predict virological and immunological control of HIV-1 infection in adolescents. AIDS 2002; 16:2275–2284.
21. Wang C, Song W, Lobashevsky E, et al. Cytokine and chemokine gene polymorphisms among ethnically diverse North Americans with HIV-1 infection. J Acquir Immune Defic Syndr 2004; 35:446–454.
23. Tang J, Naik E, Costello C, et al. Characteristics of HLA class I and class II polymorphisms in Rwandan women. Exp Clin Immunogenet 2000; 17:185–198.
24. Yunis EJ, Larsen CE, Fernandez-Vina M, et al. Inheritable variable sizes of DNA stretches in the human MHC: Conserved extended haplotypes and their fragments or blocks. Tissue Antigens 2003; 62:1–20.
25. Just JJ, King MC, Thomson G, et al. African-American HLA class II allele and haplotype diversity (corrected and republished article originally printed in Tissue Antigens 1996, Dec; 48(6):636–644). Tissue Antigens 1997; 49:547–555.
26. Cao K, Hollenbach J, Shi X, et al. Analysis of the frequencies of HLA-A, B, and C alleles and haplotypes in the five major ethnic groups of the United States reveals high levels of diversity in these loci and contrasting distribution patterns in these populations. Hum Immunol 2001; 62:1009–1030.
27. Klitz W, Maiers M, Spellman S, et al. New HLA haplotype frequency reference standards: High-resolution and large sample typing of HLA DR-DQ haplotypes in a sample of European Americans. Tissue Antigens 2003; 62:296–307.
28. Louie LG, Wallenstein E, Schmelzer K, et al. Report of the anthropology group from the Cytokine Polymorphism Component. In: Hansen J, Dupont B, eds. HLA 2004: Immunobiology of the Human MHC. Proceedings of 13th International Histocompatibility Workshop and Congress. Vol. 2. Seattle: IHWG Press, 2004.
29. Wang C, Tang J, Song W, et al. HLA and cytokine gene polymorphisms are independently associated with responses to hepatitis B vaccination. Hepatology 2004; 39:978–988.
30. Morahan G, Boutlis CS, Huang D, et al. A promoter polymorphism in the gene encoding interleukin-12 p40 (IL12B) is associated with mortality from cerebral malaria and with reduced nitric oxide production. Genes Immun 2002; 3:414–418.
31. Crowley-Nowick PA, Bell MC, Brockwell R, et al. Rectal immunization for induction of specific antibody in the genital tract of women. J Clin Immunol 1997; 17:370–379.
32. Rohan LC, Edwards RP, Kelly LA, et al. Optimization of the weck-Cel collection method for quantitation of cytokines in mucosal secretions. Clin Diagn Lab Immunol 2000; 7:45–48.
33. Hildesheim A, McShane LM, Schiffman M, et al. Cytokine and immunoglobulin concentrations in cervical secretions: Reproducibility of the Weck-cel collection instrument and correlates of immune measures. J Immunol Methods 1999; 225:131–143.
34. Crowley-Nowick PA, Ellenberg JH, Vermund SH, et al. Cytokine profile in genital tract secretions from female adolescents: Impact of human immunodeficiency virus, human papillomavirus, and other sexually transmitted pathogens. J Infect Dis 2000; 181:939–945.
35. Wang C, Tang J, Crowley-Nowick PA, et al. Interleukin (IL)-2 and IL-12 responses to Chlamydia trachomatis
infection in adolescents. Clin Exp Immunol 2005; 142:548–554.
36. Shrier LA, Bowman FP, Lin M, et al. Mucosal immunity of the adolescent female genital tract. J Adolesc Health 2003; 32:183–186.
37. Hoffmann SC, Stanley EM, Cox ED, et al. Association of cytokine polymorphic inheritance and in vitro cytokine production in anti-CD3/CD28-stimulated peripheral blood lymphocytes. Transplantation 2001; 72:1444–1450.
38. Smith KA. Interleukin-2: Inception, impact, and implications. Science 1988; 240:1169–1176.
39. Feghali CA, Wright TM. Cytokines in acute and chronic inflammation. Front Biosci 1997; 2:d12–d26.