CD4+ lymphocytes and gamma interferon predominate in local immune responses in early experimental syphilis

Abstract

The clearance of Treponema pallidum subsp. pallidum from early syphilis lesions involves infiltration of a large number of mononuclear cells and is characteristic of a cell-mediated immune response. In the present study, we sought to determine the relative abundance of different T-lymphocyte populations and Th1/Th2-associated cytokines present in testicular lesions following experimental infection with the Chicago strain of T. pallidum. Using flow cytometry, we examined the proportion of CD4(+) and CD8(+) T cells present throughout the progression and resolution of primary syphilis in the rabbit model. We related these findings to the results of real-time reverse transcription-PCR quantification of treponemal and cytokine mRNA levels. Treponemal mRNA levels reached peak values on day 18 postinfection, coincident with an initial peak in the level of T cells, which were primarily CD4(+) T cells. T-cell levels increased again during resolution of orchitis, and there was an increased proportion of CD8(+) T cells. The maximum gamma interferon (IFN-gamma) and interleukin-10 (IL-10) mRNA levels were observed on days 11 and 18, respectively, while only negligible amounts of IL-4 and IL-2 were detected throughout the infection. In addition to showing the temporal relationship between treponemal burden and T-cell responses during lesion progression, our results also demonstrate that the composition of the T-cell population changes during lesion resolution. The presence of the mRNA for IFN-gamma, but not IL-4, is consistent with cytokine expression in human syphilis and provides further support for the hypothesis that there is a Th1 predominance during the early immune response to T. pallidum.

FIG. 1.

Treponemal burden and T-lymphocyte levels in early lesions during experimental syphilis. Day 0 represents uninfected rabbits. (A) Total RNA was extracted from the testes of four animals at each time point during infection, and the levels of the 47-kDa treponemal lipoprotein (TpN47) mRNA were determined using the real-time RT-PCR relative quantification method. Data were normalized using the eukaryotic HPRT housekeeping gene. The values are the means ± standard errors of triplicate reactions for two separate tissue samples from four separate animals per time point. (B) Total cells were extracted from the testes and stained for flow cytometry using anti-rabbit PanT (Ken5) antibody for T lymphocytes as described in Materials and Methods. The values are means ± standard errors of the percentage of total acquired cells that positively stained with the anti-rabbit PanT (Ken5) antibody for four animals per time point.

FIG. 2.

CD4⁺ and CD8⁺ T-cell responses during early experimental syphilis. Day 0 represents uninfected rabbits. Total cells were extracted from the testes and stained for flow cytometry using an anti-rabbit PanT (Ken5) antibody in conjunction with either anti-rabbit CD4 or anti-rabbit CD8 as described in Materials and Methods. The values are means ± standard errors. (A) Percentage of total acquired cells that double stained for four animals per time point. (B) CD8/CD4 ratios for each of four animals per time point.

FIG. 3.

Cytokine levels during early experimental syphilis. Day 0 represents uninfected rabbits. Total RNA was extracted from the testes of four animals per time point, and IFN-γ, IL-2, and IL-4 levels (A) or IL-10 levels (B) were determined using the real-time RT-PCR relative quantitation method. Data were normalized using the eukaryotic HPRT housekeeping gene. The values are the means ± standard errors from triplicate reactions for two separate tissue samples from four separate animals per time point.