Exp Mol Pathol. 2013 Oct;95(2):166-73.

Balance of apoptotic and anti-apoptotic marker and perforin granule release in squamous intraepithelial lesions. HIV infection leads to a decrease in perforin degranulation.

Fernandes AT, da Rocha NP, Avvad E, Grinsztejn BJ, Russomano F, Tristão A, Quintana Mde S, Perez MA, Conceição-Silva F, Bonecini-Almeida Mda G.

Laboratory of Immunology and Immunogenetic in Infectious Diseases at Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.



Cell-mediated cytotoxicity plays an important role in the regulation to HPV-associated cervical intraepithelial neoplasia. HIV co-infection is related to poorer prognosis and more rapid clinical progression to cancer. We evaluated the presence of cervical inflammatory cells, apoptotic (Bax, Bcl-2, FasL, NOS2, perforin) markers and the degranulating expressing cell marker (CD107a) in low and high squamous intraepithelial lesions (LSIL and HSIL, respectively) from HIV-negative and -positive women. Higher percentage of cervical CD4+, CD8+ T cells and macrophage were observed in LSIL and HSIL groups when compared with control, especially in epithelium and basal layer of epithelium. However, progression from LSIL to HSIL did not change the frequency of inflammatory cells. HIV-infection lead to a reduction on cervical CD4+ T cell infiltration and an increased CD8+ T cell distribution in LSIL groups. A balance between pro- and anti-apoptotic protein expressions was verified. Bax-expressing cells were present in all groups and were rarely expressed in keratinocytes in the epithelium in LSIL and control groups, but notably decreased in HSIL group. However, its frequency was enhanced in the basal layer of the epithelium meanly in LSIL group. Bcl2-expressing cells in the epithelium and the stroma were enhanced in HSIL group when compared with LSIL group. HIV-infection did not interfere in both expressions NOS2 expression was located on keratinocytes in both LSIL and HSIL groups when compared with control group. There were few FasL cervical expressing cells in all groups. Indeed, perforin was identified in fewcervical cells. However, CD107a, a surfacemarker for cellular degranulationwas significantly higher in epithelium, basal layer of epithelium and stroma in LSIL and HSIL, respectively, when compared with control group. These results support that HIV infection may induce reduction on inflammatory cervical cell degranulation corroborating to carcinogenesis process. This is the first description on the role of HIV in downregulation of perforin degranulation in the cervical lesions and it might be related to carcinogenesis.

Published by Elsevier Inc.

KEYWORDS: Apoptosis, CTL, Cytotoxic dysfunction, HIV-infection, SIL, Uterine cervix

PMID: 23791892



Cervical cancer was declared the 4th cause of cancer death in 2010, and the second most common cause of cancer death in women. Infection with oncogenic types of HPV is the main causal factor for cervical cancer and its precursor lesion, squamous intraepithelial lesions (SIL). Many women are infected with HPV, but only a minority will develop SIL or cervical cancer. The majority of HPV infections induces low grade squamous intraepithelial lesion (LSIL) that in more than 90% of cases spontaneously regress and in about 10% eventually progress to high grade lesions (HSIL) and 1% frequently evolve to invasive cancer.

The mechanisms that control transition from latent infection to cervical cancer are unknown, although it is believed that progression to cervical cancer is characterized by: increased expression of viral oncoproteins E6 and E7 of high risk HPVs that deregulate the host cell growth cycle by binding and inactivating tumor suppressor proteins, cell cyclins, and cyclin-dependent kinases; integration of viral DNA into host genome, with disruption of E2 viral genes and host chromosomal loci; HIV co-infection; and cellular immunity that plays an important role in HPV infection, especially cytotoxic T cells.

Mechanisms of apoptosis are crucial for carcinogenesis control. Generally, apoptosis signaling is largely classified by the triggers involved into two pathways: the extrinsic, characterized by death receptor, as Fas and TNFR; and the intrinsic, characterized by mitochondrial dysfunction that is induced through the transcriptional or post translational regulation of apoptotic factors. The bcl-2 gene family acts as one of regulators of the apoptotic intrinsic pathway. The two most important apoptosis regulating proteins of this family are most likely a Bax and Bcl2, an apoptotic and antiapoptotic markers.

The central goal of this study was to identify cytotoxic as well as apoptotic markers in cervical uterine lesions from HPV and HPV/HIV co-infected women. Sixty women with SIL were consecutively enrolled from two cohorts from Fiocruz Clinical Care Units at Rio de Janeiro, Brazil. Cervical biopsies were snap-frozen in liquid nitrogen and mounted in OCT compound and serial cryostat sections were taken for immunohistochemistry. To determine the possible cytotoxic role of inflammatory cells, we analyzed the biomarkers FasL, perforin, CD107 (degranulation marker), Bax and Bcl2 in cervical cells by immunohistochemistry.

Balance of apoptotic and anti-apoptotic markers in HPV-infected keratinocytes determine the cervical lesion progression. So, we observed a decreased proapoptotic Bax-expressing cells in patients with more severe cervical lesion (HSIL) when compared with patients with benign lesions (LSIL) and women with no HPV lesions. Otherwise, anti-apoptotic Bcl2-expressing cells were observed meanly in HSIL women when compared with LSIL and control group (Figure 1). HIV infection do not interfered with these apoptotic markers expression. These results support an idea that HPV modulated to its favor the expression of proapoptotic and apoptotic markers in keratinocytes inducing pre-cancer lesions and further the carcinogenesis. These proteins can be used as prognostic biomarkers for pre-malign cervical lesion outcome.

Macrophages were the major inflammatory cervix cells during HPV and HIV infection. These cells produce nitric oxide from an induced nitric oxide synthase (NOS2) enzyme, an important microbicidal product. Interestingly, NOS2 expression was seen in keratinocytes located in the basal line of epithelium, instead macrophages in both LSIL and HSIL groups. This biomarker was highly expressed in both patients groups, meanly in LSIL, when compared with control group that rarely expressed NOS2. HIV-infection did not change NOS2 expression profile in the lesion (Figure 2a). The progression to more severe lesions reflect the loss of this important cellular regulatory factor. We are working to determine its role in carcinogenesis. However, NOS2 can not be used as a biomarker alone because it is induced by several inflammatory mediators during bacterial and viral infections.

Cytotoxic CD8 T cells are able to control viral infection through Fas and Perforin-based pathways, accounted for most of T-cell-mediated cytotoxicity. In cervical HPV-induced lesions, however, few cervical FasL-expressing cells in both LSIL and HSIL were observed (data not shown). Similarly, perforin expression was rare in both groups. Perforin is secreted after cytotoxic CD8 T cells encountered theirs viral targets. For monitoring the exocytosis of lytic granules in CD8+ T cells, the expression of CD107a was analyzed (Figure 2b). However, this marker was not present in the majority of inflammatory cervical cells. CD8+ T cells might have a dysfunction in the cervix, leading to fail to recognize HPV infected keratinocytes and further release of lytic granules. So, suggesting that HPV can somehow avoid the induction of an effective cellular immunity and escape of immune surveillance, leading to cervical lesion progression and further cancer development.

We believe that the cervical inflammatory microenvironment induced by HPV infection and host immune response can define the lesion progression. More studies are needed to determine prognostic biomarkers and to identify viral and host targets for new drugs discover.
figure-1Figure 1. Distribution of Bax- (A) and Bcl2-expressing cells (B) in uterine cervix from Control, LSIL and HSIL patients. The number of positive cells was demonstrated in epithelium, basal layer of epithelium , stroma and periglandular area.

Figure 2. Distribution of NOS2- (A) and CD107-expressing cells (B) in uterine cervix from Control, LSIL and HSIL patients. The number of positive cells was demonstrated in epithelium, basal layer of epithelium, stroma and periglandular area.

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