SYPHILIS CONTINUES TO be a significant worldwide venereal disease as a result of Treponema pallidum (TP) infection. Syphilitic lesions are infiltrated by CD4+, CD8+ T lymphocytes and macrophages, and the major mechanism of TP clearance is that activated macrophages phagocytose opsonized TPs. 1,2 Syphilitic patients have decreased CD4+ cells and increased CD8+ cells in peripheral blood. 3 At present, however, the relative roles of humoral and cellular immunity in protection against syphilis remain an enigma. Little is also known about what enables TPs to escape the robust immune responses, invade almost all tissues, and persist for a long time. 4,5
Fitzgerald 6 provided a hypothesis in the rabbit model that a Th1 response is dominant in primary syphilis, and then shifts to a Th2 response as the disease progresses to the secondary stage. In addition, the cytokine gene expression in skin of C4-deficient guinea pigs infected with TP showed a local Th2 response being consistent with an adequate immune response. 7 Nevertheless, Arroll et al. 1 observed that the general T-cell response to TP antigens in syphilis infection was biased toward the Th1 phenotype; Podwinska et al. 8 believed that such shift to Th2 could occur in early latent syphilis, and van Voorhis et al. 2 argued against a Th1 to Th2 shift from the primary to the secondary stage.
Recently, the relationship between apoptosis and microbial infection has become the focus of scientists’ attention. Apoptosis, or programmed cell death, is a physiological cell death process that is not only crucial for the proper development and homeostasis of many tissues, but also plays an important role in the regulation of immune responses. 9,10 Bacterium, parasite, and virus infections can modulate apoptosis; of them, HIV infection is studied mostly. CD4+ and CD8+ T cells of HIV-infected subjects show an increased susceptibility to spontaneous and activation-induced apoptosis. 9–11 Nevertheless, there is a complex balance between bacterium infection and apoptosis, and the molecular mechanisms of bacterial-associated apoptosis are incompletely clearcut. Bacteria or parasites induce apoptosis in immune cells responding to infection to inhibit an immune response, or inhibit apoptosis in directly infected cells to facilitate intracellular replication or survival. 9
However, whether syphilis influences lymphocyte apoptosis has not been reported so far. We detected the peripheral blood Th and Tc subsets in secondary early syphilis using 3-color flow cytometry, and found that Th1 cells and ratios of Th1/Th2 and Tc1/Tc2 in syphilitics were obviously decreased, and Tc0 and Tc2 cells were significantly increased in syphilitics as compared with control subjects (data not shown). Based on the aforementioned studies, we hypothesize that peripheral blood lymphocyte (PBL) apoptosis is involved in the dysregulation of cellular immune responses in secondary early syphilitics, and its molecular mechanism is implicated in the abnormal expression of Fas and Bcl-2. To test this hypothesis, we examined PBL immunophenotype, apoptosis, and expression of Fas (CD95) and Bcl-2 by flow cytometry.
Materials and Methods
Thirty-three patients with secondary early syphilis, 20 men and 13 women, aged 20 to 42 years (mean age, 28 years) were enrolled from the Dermato-Venereal Clinic of Affiliated Hospital of Guangdong Medical College between 2001 and 2002. Diagnosis was based on a compatible history, clinical examination, and the results of nontreponemal and treponemal serologic tests. No patients received antibiotic treatment, had a positive history of autoimmune diseases, or were HIV-seropositive. Thirty healthy volunteers, 18 males and 12 females, aged 18 to 40 years (mean age, 26 years) served as the control group.
Antibodies and Reagents
Antihuman CD4-PE, CD8-PE, CD19-PE and mouse IgG1-PE, and RBC Lysis Buffer were products of eBioscience (USA). Antihuman CD95-FITC, Bcl-2-FITC, and mouse IgG1-FITC were obtained from Ancell Corporation (USA). Annexin V-FITC Apoptosis Detection Kit and lymphocyte separation solution were purchased from BioVision (USA) and Beijing TBD Biologic Technology Ltd. (China), respectively.
Detection of Lymphocyte Immunophenotypes
One hundred microliters of heparinized peripheral whole blood were gently mixed with 10 μL of PE-labeled monoclonal antibodies (CD4, CD8, CD19, or negative control IgG1) and incubated 30 minutes at room temperature in the dark. After adding RBC lysis buffer for 10 minutes, the samples were centrifuged at 1500× g for 5 minutes and the supernatant was discarded. The samples were washed with 2 mL phosphate-buffered saline (PBS), centrifuged, and the supernatant was discarded. The cells were stored in 1% paraformaldehyde in PBS (500 μL) at 4°C until acquisition within 4 hours.
Heparinized peripheral blood (5 mL) was diluted with 1:2 lymphocyte separation solution. PBLs were obtained by density gradient separation and then washed twice with Hanks’ balance salt solution (HBSS). The purified population was generally more than 95% lymphocytes.
Apoptosis Detection With Annexin V-FITC Staining
PBL apoptosis was detected with annexin V (AV)-FITC staining according to the manufacturer’s instruction. Briefly, resuspended PBLs at a density of 1 × 106 cells/mL in 500 μL of 1X× binding buffer were incubated with 10 μL of PE-conjugated monoclonal antibodies (CD4, CD8, or CD19), 5 μL of AV-FITC, and 5 μL of propidium iodide (PI) for 20 minutes at 4°C in the dark. IgG1-PE and IgG1-FITC antibodies were used as a negative control. The samples were washed twice in PBS and then stored in 1% paraformaldehyde in PBS (500 μL) at 4°C until acquisition within 4 hours.
Determination of Fas and Bcl-2 Expression
The resuspended PBLs at a density of 1 × 106 cells/mL in 200 μL of 1X × binding buffer were incubated with CD4-PE (10 μL), CD95-FITC (10 μL), CD4-PE (10 μL), Bcl-2-FITC (10 μL), CD8-PE (10 μL), CD95-FITC (10 μL), CD8-PE (10 μL), Bcl-2-FITC (10 μL), CD19-PE (10 μL), CD95-FITC (10 μL), CD19-PE (10 μL), Bcl-2-FITC (10 μL) for 30 minutes at room temperature in the dark. IgG1-PE and IgG1-FITC antibodies were used as a negative control. The samples were washed twice in PBS and then stored in 1% paraformaldehyde in PBS (500 μL) at 4°C until acquisition within 4 hours. It was noticeable that these reagent incubations and washes for Bcl-2 staining were done using a buffer containing 0.3% saponin to permeabolize cells because of antibody Bcl-2 recognizing the intracellular Bcl-2 protein.
Samples were analyzed on a Coulter Epics-XL flow cytometer (USA). Two-parameter histograms were created by using CELLQUEST software. A total of 10,000 lymphocytes were counted in each sample. Cells were illuminated with a 488-nm wavelength argon ion laser using a standard filter combination. Lymphocytes were gated out or included for assay according to lightscatter data, forward lightscatter measuring size, and side lightscatter measuring granularity or density. AV+PI− cells and AV+PI+ cells were defined as apoptotic cells and necrotic cells, respectively.
Results for positive cells (mean ± standard deviation) were expressed as a percentage of the respective subpopulation. Intergroup comparisons and correlation coefficients were performed by Student t test and Pearson’s correlation analysis, respectively. P <0.05 was considered significant.
Peripheral Blood Lymphocyte Subpopulations
The percentage of CD4+ cells and the ratio of CD4+:CD8+ were significantly decreased, and that of CD8+ cells was obviously increased in syphilitic patients as compared with the control subjects, but there was no statistical difference in that of CD19+ cells (B lymphocytes) between 2 groups (Table 1).
Peripheral Blood Lymphocyte Apoptosis and Necrosis
The apoptosis and necrosis percentage of PBLs and CD4+ cells was significantly higher in the syphilitic group than that in the control group, but there was no difference in that of CD8+ and CD19+ cells between them (Table 2).
Fas and Bcl-2 Expression in Peripheral Blood Lymphocyte Subpopulations
The percentage of CD95+ PBLs and CD95+ CD4+ cells was significantly higher, and that of Bcl-2+ PBLs and Bcl-2+ CD4+ cells was remarkably lower in the syphilitic group than those in the control group, but there was no difference in that of CD95+ CD8+ and Bcl-2+ CD8+ cells, and CD95+ CD19+ and Bcl-2+ CD19+ cells between them (Table 3).
Relationship Between Expression of Fas and Bcl-2 and Apoptosis in Peripheral Blood Lymphocyte Subpopulations in Syphilitics
The percentage of PBLs and CD4+ cells undergoing apoptosis correlated positively with Fas expression (r = 0.685, P <0.01; r = 0.712, P <0.01) and negatively with Bcl-2 expression (r = −0.824, P <0.01; r = −0.736, P <0.01), but there were no correlations between that of CD8+ and CD19+ cells undergoing apoptosis and their expression of Fas and Bcl-2 (P >0.05).
CD4+ and CD8+ T cells, commonly known as Th and Tc cells, respectively, play important immunoregulatory and effector roles in immune responses. CD4+ T cells promote antibody responses and mediate delayed-type hypersensitivity (DTH) and are required for CD8 response, whereas CD8+ T cells are pivotal in the control of viral infections, and many nonviral intracellular pathogens by killing the infected targets and also mediate inflammation. In addition, CD4+ T cells mediate cytolysis, whereas CD8+ T cells induce similar DTH. 12,13 CD8+ T cells also regulate the activation and differentiation of CD4+ cells, the development of CD4 perforin-mediated cytotoxicity, suppress CD4 proliferative responses, and influence other components of the immune response such as eosinophil recruitment, macrophage activation, and antibody production by B cells. 14 As compared with the control subjects, the percentage of CD4+ T cells and the ratio of CD4+:CD8+ were significantly decreased, and that of CD8+ T cells was obviously increased in syphilitic patients, but there was no statistical difference in that of CD19+ cells between the 2 groups. Our results are in agreement with Pope’s report, 3 suggesting patients with secondary early syphilis have a dysregulation of cellular immunity.
At the earlier stage of the adaptive immune response, professional antigen-presenting cells (APCs) present pathogen antigens in the context of MHC II molecules to antigen-specific CD4+ T cells. Then, CD4+ T cells provide help for CD8+ and B cell responses by releasing different cytokines. This help is especially crucial for the activation and expansion of naive B and T lymphocytes. 12 Meanwhile, CD8+ T cells can alter the balance of Th1/Th2 responses in vivo, and the exacerbated CD8+ T cell responses are capable of leading to potential damage for both cell killing and inflammation. 13,14 Tc2 cells can promote Th2 effectors as well as suppress the development of Th1 cells. 14 Furthermore, CD25+ CD4+ regulatory T cells can control and eventually suppress autoreactive T cells or harmful T cells activated during normal immune responses, and the elimination or inactivation of these regulatory T cells is associated with the onset of autoimmune and inflammatory diseases and with abnormal peripheral T cell homeostasis. 15 Therefore, decreased amounts of CD4+ T cells could lessen the DTH against TP and the important cell-cell interaction and cause immune dysfunction, whereas increased CD8+ T cells could uncertainly strengthen the capacity of cytolysis. This disturbance of cell-mediated immunity might contribute to the incomplete clearance of TP from the lesions and the chronic infection.
AV is a Ca2+-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS). The translocation of PS from the inner side of the plasma membrane to the outer layer is the early alteration of apoptotic cells, but this change also occurs during cell necrosis. PI staining is widely used to discriminate living cells from dead cells. Therefore, the measurement of AV binding to the cell surface as indicative for apoptosis has to be made in combination with a dye exclusion test such as PI staining to determine the integrity of the cell membrane. This method is sensitive and easy to perform, and capable of detecting early phases of apoptosis. 16,17 The combined use of FITC-labeled AV and dye exclusion of PI in the current study can discriminate the intact cells (AV−PI−), apoptotic cells (AV+PI−) and necrotic cells (AV+PI+). The apoptotic and necrotic rates of PBLs and CD4+ T cells in freshly isolated PBLs were significantly increased in syphilitics as compared with control subjects. The results presented here testify to our hypothesis that PBL apoptosis is involved in the dysregulation of cellular immune responses in secondary early syphilitics and, at least partly, could explain the cause of decreased amounts of CD4+ T cells.
Many molecules transduce apoptotic signals, and certain types of apoptosis originate from the ligation of death receptors. Fas (CD95), a member of the tumor necrosis factor (TNF) receptor superfamily expressed on various tissues, is the best characterized death receptor and its signaling is necessary for a variety of immunologic processes. 9 T cells expressing CD95 are increasingly susceptible to anti-CD95-mediated apoptosis. 18 The Bcl-2 family members are cytoplasmic regulators of apoptosis; of them, Bcl-2 inhibits apoptosis by enhancing cell survival rather than by accelerating cell proliferation rate. 9,10
Although alterations in death receptors or specific death receptor ligands, caspases, and the Bcl-2 family have all been reported to regulate the apoptosis of CD4+ T cells in HIV infection, enhanced Fas-mediated apoptosis might be an important mechanism. 9–11 TNF receptor-mediated cell death of T cells in HIV-infected subjects is associated with both alteration of Bcl-2 expression and activation of caspase-3 and caspase-8. 19 In HIV-infected subjects, IL-2-producing T cells have an increased susceptibility to apoptosis, which is related to a downregulation of Bcl-2 expression 11; IL-15 mediates its apoptosis-blocking effects by suppressing the downmodulation of Bcl-2. 10 There are at least 3 different mechanisms responsible for apoptosis of in vitro-cultured T cells from HIV-infected patients: spontaneous cell death which is CD95-independent, CD95-induced cell death, and activation-induced cell death (AICD) by CD95-dependent and -independent pathways. 20 In addition, HSV-1 enhances apoptosis of CD4+ and HLA-DR+ T cells, but not of CD8+ T cells, whereas Bcl-2 does not contribute to HSV-1-induced T lymphocyte apoptosis. 21
To determine whether increased PBL apoptosis in secondary syphilis is associated with the abnormal expression of Fas and Bcl-2, we compared PBL Fas and Bcl-2 expression in syphilitics and control subjects, and analyzed the relationship between expression of Fas and Bcl-2 and apoptosis in PBLs of syphilitics. As compared with the control group, Fas overexpression and Bcl-2 downexpression in the syphilitic group were seen in PBLs and CD4+ cells but not in CD8+ and CD19+ cells. PBL and CD4+ cell apoptosis correlated positively with Fas expression and negatively with Bcl-2 expression in syphilitics. The present results indicate that the Fas-mediated death pathway and antiapoptotic Bcl-2 protein are both responsible for PBL and CD4+ cell apoptosis. However, it is unknown why CD8+ and CD19+ cells do not exhibit apoptosis. The possible mechanisms are as follows: first, in view of Th1 and Tc2 cells being more sensitive to apoptosis than Th2 and Tc1 cells, respectively, 11,14 various lymphocytes could have different susceptibility of apoptosis. Second, the expression of CD95 alone is not sufficient for apoptotic susceptibility, and other components that regulate the Fas-dependent apoptosis could contribute to the protection of CD8+ and CD19+ cells. 9,14,18 Finally, there could exist the overexpression of other members of the antiapoptotic Bcl-2 family and other inhibitors of apoptosis.
In conclusion, the increased apoptosis of PBLs and CD4+ T cells by the Fas-mediated death pathway and downexpression of Bcl-2 protein could account for dysfunction of cellular immunity in secondary early syphilis, which is responsible for the incomplete clearance of TP from the lesions and the chronic infection.
1. Arroll TW, Centurion-Lara A, Lukehart SA, et al. T-cell responses to Treponema pallidum subsp. pallidum antigens during the course of experimental syphilis infection. Infect Immun 1999; 67: 4757–4763.
2. van Voorhis WC, Barrett LK, Koelle DM, et al. Primary and secondary syphilis lesions contain mRNA for Th1 cytokines. J Infect Dis 1996; 173: 491–495.
3. Pope V, Larsen SA, Rice RJ, et al. Flow cytometric analysis of peripheral blood lymphocyte immunophenotypes in persons infected with Treponema pallidum. Clin Diagn Lab Immunol 1994; 1: 121–124.
4. Morgan CA, Lukehart SA, van Voorhis WC. Immunization with the N-terminal portion of Treponema pallidum repeat protein K attenuates syphilitic lesion development in the rabbit model. Infect Immun 2002; 70: 6811–6816.
5. Morgan CA, Molini BJ, Lukehart SA, et al. Segregation of B and T cell epitopes of Treponema pallidum repeat protein K to variable and conserved regions during experimental syphilis infection. J Immunol 2002; 169: 952–957.
6. Fitzgerald TJ. The Th1/Th2-like switch in syphilitic infection: is it detrimental? Infect Immun 1992; 60: 3475–3479.
7. Wicher V, Scarozza AM, Ramsingh AI, et al. Cytokine gene expression in skin of susceptible guinea-pig infected with Treponema pallidum. Immunology 1998; 95: 242–247.
8. Podwinska J, Lusiak M, Zaba R, et al. The pattern and level of cytokines secreted by Th1 and Th2 lymphocytes of syphilitic patients correlate to the progression of the disease. FEMS Immunol Med Microbiol 2000; 28: 1–14.
9. Dockrell DH. Apoptotic cell death in the pathogenesis of infectious diseases. J Infect 2001; 42: 227–234.
10. Chang KH, Kim JM, Kim HY, et al. Spontaneous programmed cell death of peripheral blood mononuclear cells from HIV-infected persons is decreased with interleukin-15. Yonsei Med J 2000; 41: 112–118.
11. Ledru E, Lecoeur H, Garcia S, et al. Differential susceptibility to activation-induced apoptosis among peripheral Th1 subsets: Correlation with Bcl-2 expression and consequences for AIDS pathogenesis. J Immunol 1998; 160: 3194–3206.
12. Johnson RM, Brown EJ. Cell-mediated immunity in host defense again infectious diseases. In: Mandell GL, Bennet JE, Dolin R, eds. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, 5th ed. Beijing: Science Press, 2001: 112–146.
13. Sad S, Li L, Mosmann TR. Cytokine-deficient CD8+ Tc1 cells induced by IL-4: Retained inflammation and perforin and Fas cytotoxicity but compromised long term killing of tumor cells. J Immunol 1997; 159: 606–613.
14. Vukmanovic-Stejic M, Vyas B, Gorak-Stolinska P, et al. Human Tc1 and Tc2/Tc0 CD8 T-cell clones display distinct cell surface and functional phenotypes. Blood 2000; 95: 231–240.
15. Banz A, Pontoux C, Papiernik M. Modulation of Fas-dependent apoptosis: A dynamic process controlling both the persistence and death of Cd4 regulatory T cells and effector T cells. J Immunol 2002; 169: 750–757.
16. Vermes I, Haanen C, Steffens-Nakken H, et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Methods 1995; 184: 39–51.
17. Basco Z, Everson RB, Eliason JF. The DNA of annexin V-binding apoptotic cells is highly fragmented. Cancer Res 2000; 60: 4623–4628.
18. McCloskey TW, Bakshi S, Than S, et al. Immunophenotypic analysis of peripheral blood mononuclear cells undergoing in vitro apoptosis after isolated from human immunodeficiency virus-infected children. Blood 1998; 92: 4230–4237.
19. de Oliveira Pinto LM, Garcia S, Lecoeur H, et al. Increased sensitivity of T lymphocytes to tumor necrosis factor receptor 1 (TNFR1)- and TNFR2-mediated apoptosis in HIV infection: Relation to expression of Bcl-2 and active caspase-8 and caspase-3. Blood 2002; 99: 1666–1675.
20. Bohler T, Debatin KM, Linde R. Sensitivity of CD4+ peripheral blood T cells toward spontaneous and CD95 (APO-1/Fas)-induced apoptosis in pediatric human immunodeficiency virus infection. Blood 1999; 94: 1829–1833.
21. Ito M, Watanabe M, Kamiya H, et al. Herpes simplex virus type 1 induces apoptosis in peripheral blood T lymphocytes. J Infect Dis 1997; 175: 1220–1224.