Analysis of Cytokine Profiles in Patients With Human Papillomavirus-Associated Neoplasms

Cervical cancer is the second leading cause of cancer mortality in women worldwide, with approximately half a million new cases occurring annually ( 1 ). More than 90% of cervical cancers and their precursors, so-called cervical intraepithelial neoplasia, contain human papillomavirus (HPV) DNA sequences, and it is now well established that HPV has a major causal role in the development of cervical neoplasia ( 2 ). It has also been recognized that HPV infection of the cervix and lower genital tract is one of the most common sexually transmitted diseases ( 3 ). Prevalence rates of HPV infection (based on nucleic acid amplification techniques) can be only crudely estimated because they are influenced by several factors. These rates vary substantially according to the population studied and are highly age dependent. In the United States, HPV prevalence ranges from 25%-30% in asymptomatic women aged 15-25 years to approximately 5% in women aged 33-55 years, an age group in which most cervical cancers are detected ( 2 , 4 ). In contrast, there are approximately 15 000 incident cases of cervical cancers in the United States annually ( 5 ). These observations have led investigators to postulate that other factors, in addition to HPV, play a role in cervical carcinogenesis. Several lines of evidence suggest that cell-mediated immune responses are important in controlling both HPV infections and HPV-associated neoplasms [for review, see ( 6 )]. First, the prevalence of HPV-related diseases (infections and neoplasms) is increased in transplant recipients and in patients infected with human immunodeficiency virus (HIV); both types of patients are known to have impaired cell-mediated immunity ( 7 , 8 ). Second, studies on animals have demonstrated that immunized animals are protected from papillomavirus infection and from the development of neoplasia. Immunization also facilitates the regression of existing lesions ( 9 – 11 ). Third, infiltrating CD4 + (T-helper cells) and CD8 + (cytotoxic/suppressor T cells) T cells have been observed in spontaneously regressing warts. Fourth, warts seen in patients who are on immunosuppressive therapy often disappear when this treatment is discontinued. Cell-mediated immunity is regulated by cytokines that are secreted by T helper cells. In general, T-helper cells can be classified as Th1 and/or Th2 cells on the basis of the different types of cytokines they secrete. Th1 cells secrete interleukin (IL) 2 (IL-2) and interferon gamma (IFN γ). Th2 cells produce IL-4, IL-5, IL-10, and IL-13. The Th1 lymphocytes are the most important effector cells in inflammatory reactions associated with vigorous delayed-type hypersensitivity but low antibody production, as occurs in contact dermatitis and in viral or intracellular bacterial infections [for review, see ( 12 , 13 )]. The functional phenotype of most Th2 cells may account for both the persistent production of certain antibody isotypes, particularly immunoglobulin G 1 and immunoglobulin E, and the eosinophilia observed in human helminthic infections and allergic disorders. Although the Th1 and Th2 phenotypes were first described in mice, clones of Th1 and Th2 cells have also been isolated from humans. For example, most CD4 + T cells that infiltrate the thyroid gland in patients with autoimmune thyroid diseases are of the Th1 type, since they can be induced to produce IFN γ but not IL-4. In contrast, the great majority of allergen-specific T-cell clones derived from patients with allergic disorders express the Th2 phenotype, as evidenced by their production of IL-4 and IL-5 and their limited, if any, production of IFN γ ( 13 ). Lymphocyte- mediated protection from viral infections as well as control of tumors is thought to be mediated by Th1 cytokine responses and impaired by Th2 cytokine responses. The IL-2- and IFN γ-producing Th1 response is likely to be the major component that contributes to the development of cell-mediated immunity against HPV infections and HPV-associated neoplasms. In this issue of the Journal, Clerici et al. ( 14 ) report on the cytokine profiles of stimulated peripheral blood mononuclear cells (PBMCs) in women with precursors of cervical cancer (grade III cervical intraepithelial neoplasia) and HPV infection. Women with HPV infection limited to the cervix were compared with women with more extensive HPV infection that involved other sites of the lower genital tract. These investigators demonstrate that IL-2 production by PBMCs, in response to stimulation with soluble antigen or alloantigens (i.e., major histocompatibility complex [MHC] antigens), was reduced in the group of patients with extensive HPV infection compared with the group of patients with localized disease and with healthy control subjects. Furthermore, IL-4 and IL-10 production in response to mitogen stimulation was elevated in the group with extensive disease compared with the response seen in the group with localized disease or healthy control subjects. The same group of investigators ( 15 ) made similar observations in an earlier study performed on asymptomatic, HIVseropositive individuals. A sequential and progressive loss of Th1 function was observed; it was manifested first by the loss of the IL-2 response to recall antigens, then by the IL-2 response to alloantigens, and finally by the IL-2 response to phytohemagglutinin (PHA). Furthermore, it was reported ( 16 ) that these defects were associated with, or predictive of, a variety of acquired immunodeficiency syndrome (AIDS)-related events. Individuals who were unresponsive to all three stimuli (recall antigens, alloantigens, and PHA) exhibited a more rapid decline in CD4 + T-cell numbers, a higher frequency of bacterial and opportunistic infections, and a greater likelihood for their disease to progress rapidly to AIDS. A similar decrease in the Th1 response with predominance of the Th2 cytokine profile has also been observed in patients with such neoplasms as Sézary syndrome ( 17 ), Hodgkin's disease ( 18 ), bronchogenic carcinoma ( 19 , 20 ), renal cell carcinoma ( 21 , 22 ), lymphomas ( 23 ), glioma ( 24 ), and melanoma ( 25 , 26 ). These observations clearly demonstrate that the Th2 phenotype is associated with persistence of viral infection and development of neoplasms. Although the study by Clerici et al. presented in this issue of the Journal did not use an HPV-specific peptide as a recall antigen, HPV antigen-specific assays were performed in a study by Tsukui et al. ( 27 ) and showed similar findings. Tsukui et al. demonstrated that Th1 cytokine production, specifically IL-2 production, in response to HPV-16 E6 and E7 peptides is diminished in patients with high-grade cervical intraepithelial lesions and cervical cancer compared with that in cytologically normal women ( 27 ). These findings are consistent with the hypothesis that the production of cytokines that enhance potentially protective cell-mediated immunity is defective in women with extensive HPV infection and that progression to cervical cancer precursor lesions may be associated with a Th1-to-Th2 switch in immunoregulatory cytokine production. At present, it is unknown whether this Th1-to-Th2 shift leads to the development of neoplasia by subverting immune surveillance mechanisms or whether the shift is a secondary effect induced by the tumor cells or by the persistent viral infection itself. One argument against the hypothesis that the shift from a Th1 to a Th2 response is the cause of the progression to neoplasia is that patients with allergic disorders, who in general have high Th2-type responses, do not appear to have higher rates of viral infections or cancer ( 28 ). Furthermore, HPV-infected epithelial cells have been shown to modify cytokine profiles. Lymphokine secretion by HPVimmortalized and cervical carcinoma cell lines has been demonstrated in culture ( 29 ). In addition, constitutive release of IL-6 by HPV-16-harboring keratinocytes ( 30 ) and enhanced IL-6 gene expression in invasive cervical carcinoma have been reported ( 31 ). These findings suggest that HPV-infected keratinocytes can modify local immune responses and possibly can influence systemic cytokine profiles. Accordingly, it will be important to conduct prospective, longitudinal studies to determine whether the shift of cytokine profiles from Th1 to Th2 is a causal factor or an epiphenomenon. It has also been shown that the pattern of cytokine profiles can be modified in vitro and in vivo. For example, IL-12 or IFN γ can decrease Th2 clones, and IL-10 can decrease Th1 clones. IL-12 has been shown to restore IFN γ production by PBMCs from HIV-seropositive patients in whom IFN g production by PBMCs was reduced or lost ( 32 ). In an in vivo murine model, treatment with recombinant murine IL-12 cured most BALB/c mice infected with Leishmania ( 33 ). Cure was associated with a markedly depressed production of IL-4 by lymph node cells cultured with antigen or mitogen, whereas IFN γ production was preserved or increased. Thus, murine IL-12 prevents deleterious Th2 T-cell responses and promotes curative Th1 responses in an IFN γ-dependent fashion during murine leishmaniasis. These findings suggest that treatment with cytokines may have potential in augmenting the diminished immunologic functions associated with progression of those diseases influenced by inappropriate cytokine production. In summary, the study by Clerici and colleagues ( 14 ) adds to our growing understanding of the mechanisms by which the immune system mediates the behavior of HPV-associated neoplasms. Further studies aimed at elucidating how cytokine production is regulated in response to HPV infection and what the role of cytokines is in the critical step between infection and neoplasia hold promise for developing novel approaches to the therapy of HPV infection and cervical cancer.