ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2000, p. 1869–1873 0066-4804/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 44, No. 7
Correlation between Pretreatment Levels of Interferon Response Genes and Clinical Responses to an Immune Response Modifier (Imiquimod) in Genital Warts I. ARANY,1* S. K. TYRING,1,2 M. M. BRYSK,1,2 M. A. STANLEY,3 M. A. TOMAI,4 R. L. MILLER,4 M. H. SMITH,4 D. J. MCDERMOTT,4 AND H. B. SLADE4 Departments of Microbiology and Immunology1 and Dermatology,2 University of Texas Medical Branch, Galveston, Texas; Department of Pathology, University of Cambridge, Cambridge, United Kingdom3; and 3M Pharmaceuticals, St. Paul, Minnesota4 Received 13 December 1999/Returned for modification 13 March 2000/Accepted 25 April 2000
Imiquimod (IQ) has been successfully used in treatment of genital warts. In clinical settings, patients responded well but wart reduction rates varied. Our aim was to find a correlation between clinical responses and pretreatment (constitutive) levels of genes that might be involved in the molecular action of IQ. Since IQ is a cytokine inducer, we analyzed levels of expression of genes of the JAK/STAT signaling pathway and their inhibitors as well as interferon response factors (IRFs) in pretreatment biopsy specimens from complete responders (99 to 100% wart reduction rate) versus incomplete responders (75 to 92% wart reduction rate) by reverse transcription-PCR. We found that mRNA levels of signal transducer and activator of transcription 1 (STAT1) and IRF1 were higher in complete responders than in incomplete responders. Incomplete responders expressed larger amounts of STAT3, IRF2, and protein inhibitor of activated STAT1 (PIAS1) mRNAs compared to complete responders before IQ treatment. We hypothesize that high-level expression of STAT1 and IRF1 is advantageous for a better IQ response. The observed differences in constitutive mRNA levels of these genes may be the consequence of alterations in cellular differentiation and/or variable expression of endogenous interferons. Previous in vitro studies showed that keratinocyte differentiation coordinates the balance between positive and negative signals along the JAK/STAT pathway by regulating the IRF1:IRF2 and STAT1: PIAS1 ratios and thus affecting induction of IQ-inducible genes. Specifically, differentiation supports constitutive expression of STAT1 and IRF1 mRNAs but not expression of IRF2 and PIAS1. Our data are in good agreement with studies that showed the importance of STAT1 in cytokine induction and activation of interferon-responsive genes by IQ. regulatory mechanisms, such as negative regulators of the JAK/STAT pathway (35). Accordingly, our aim was to determine, through post hoc evaluation after unblinding, whether clinical responses to IQ treatment correlate with pretreatment mRNA levels of STATs,
Imiquimod (IQ) is an immune response modifier (30) that is capable of inducing several cytokines, such as alpha interferon (IFN-␣), interleukin-6 (IL-6), and IL-8, in different cell types (12, 37). In keratinocytes an increase in IL-6 and IL-8 mRNA levels has been observed after IQ treatment (23). In the clinic, IQ has been successfully used for treatment of genital warts (1, 5, 10). IQ induces several cytokines, including IFN-␣, during the course of therapy (38); this is believed to play an important role in this drug’s mechanism of action (33). Also, induction of IFN and stimulation of IFN-responsive genes by IQ require active signal transducer and activator of transcription 1 (STAT1) (6). In a clinical study, we found that every patient responded to IQ therapy but that the degree of response varied (38). In a previous study we demonstrated that the extent of the IFN response depends on the differentiation status of the untreated genital warts (2) and correlates with pretreatment mRNA levels of various genes (3, 4, 38). Other studies of myeloid leukemias showed a correlation between the interferon response factor 1 (IRF1):IRF2 ratio and both the cytogenetic and molecular responses to IFN-␣ treatment (19). Recent studies revealed that the duration and intensity of a cell’s response to cytokines appear to be determined by the net effect of several
FIG. 1. Representative RT-PCR results for STAT1 in various biopsy specimens. mRNAs from biopsy specimens from complete responders (A) and incomplete responders (B) were reverse transcribed and then PCR amplified, using gene-specific primer pairs. PCR fragments were resolved on a 1.5% agarose gel in Tris-borate-EDTA. Fragments were transferred to a nylon membrane and then hybridized with gene-specific probes prior to autoradiography. This figure shows results for STAT1 as well as the constitutively expressed G3PDH. Quantitation is seen in Fig. 2.
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1070. Phone: (409) 772-8145. Fax: (409) 7476869. E-mail:
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FIG. 2. mRNA levels of STATs (A) and inhibitors of JAK/STAT signaling (B) in wart biopsy specimens before IQ treatment. Biopsy specimens from complete responders and from incomplete responders were analyzed by a semiquantitative RT-PCR method. cDNAs were obtained from the isolated RNAs by RT and subjected to PCR with gene-specific primer pairs. PCR fragments were resolved on an agarose gel and transferred to a nylon membrane. Hybridization with gene-specific probes and subsequent autoradiography revealed the identities of fragments, and quantitation was done by densitometry. Data are expressed as ratios of target gene and G3PDH mRNA levels. Data were plotted as box plots and analyzed for statistical differences. The box extends from the 5th to the 95th percentile; the horizontal line indicates the median, and the error bars show the range of the data. N.S., not significant.
IRFs, and/or negative regulators of the JAK/STAT pathway in genital wart biopsy specimens. MATERIALS AND METHODS Selection of biopsy specimens. In a randomized, double-blind, vehicle-controlled study (38), patients were given IQ in the form of a self-applicable cream (Aldara) three times per week for a maximum of 16 weeks. All patients who received Aldara, i.e., 16 of 20 patients who were enrolled in the study, responded to the treatment; the remaining 4 patients received a placebo. However, the extent of wart clearance was variable; based on the rate of clearance, patients were evaluated in two groups: complete responders (10 patients, with 99 to 100% wart reduction) and incomplete responders (6 patients, with 75 to 92% wart
ANTIMICROB. AGENTS CHEMOTHER. reduction). Biopsy specimens obtained prior to drug treatment (38) were used in these experiments. Cell cultures. Primary normal human keratinocytes were obtained from foreskin as described elsewhere (7). Cells were kept in serum-free KGM medium (Clonetics, Palo Alto, Calif.). For induction of keratinocyte differentiation, KGM medium was supplemented with 2 mM calcium (8, 17). Semiquantitation of mRNA levels. Total RNA was isolated from the biopsy specimens or cell monolayers by using Tri-Reagent (Molecular Diagnostics, Cincinnati, Ohio) in accordance with the manufacturer’s recommendation. mRNA levels of various genes were determined by a semiquantiative reverse transcription (RT)-PCR (38). One microgram of RNA was reverse transcribed (SuperScript II; GIBCO/BRL) and subjected to PCR amplification. Primer pairs were custom designed and synthesized (Genosys, The Woodlands, Tex.). The PCR run included reagent controls as well as cloned cDNAs as positive controls. PCR fragments were resolved by agarose gel electrophoresis, transferred to a Hybond N⫹ nylon membrane (Amersham, Arlington Heights, Ill.), and hybridized with end-labeled gene-specific oligonucleotide probes. Autoradiograms were analyzed by densitometry (AlphaImager; Alpha Innotech, San Leonardo, Calif.). Levels of various mRNAs were expressed as ratios of the target gene mRNA levels to that of the mRNA for the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene. Statistical analysis. Data from the two groups were compared by the t test and the Mann-Whitney rank sum test. Results are presented as box plots.
RESULTS Pretreatment mRNA levels of various STATs in wart biopsy specimens. RT-PCR studies determined the STAT1 and STAT3 mRNA levels in specimens from pretreatment biopsies of genital warts. The results of a representative experiment are shown in Fig. 1. This figure demonstrates the equal RNA loads (equal amounts of constitutive G3PDH) but uneven levels of STAT1. Densitometric analyses revealed that complete responders had significantly higher levels of STAT1 mRNA and lower levels of STAT3 mRNA than did incomplete responders (Fig. 2A). STAT2 mRNA levels were very low and were similar in the two groups (data not shown). Pretreatment mRNA levels of various inhibitors of the JAK/ STAT pathway. mRNA levels of protein inhibitor of activated STAT1 (PIAS1) were significantly higher in the incomplete responder group than in the complete responders (Fig. 2B). PIAS3 was equally expressed in the two groups. Other inhib-
FIG. 3. mRNA levels of IRFs (A) and endogenous IFNs (B) in wart biopsy specimens before IQ treatment. Biopsy specimens from complete responders and from incomplete responders were analyzed by a semiquantitative RT-PCR method. cDNAs were obtained from the isolated RNAs by RT and subjected to PCR with gene-specific primer pairs. PCR fragments were resolved on an agarose gel and transferred to a nylon membrane. Hybridization with gene-specific probes and subsequent autoradiography revealed the identities of fragments, and quantitation was done by densitometry. Data are expressed as ratios of target gene and G3PDH mRNA levels. Data were plotted as box plots and analyzed for statistical differences. The box extends from the 5th to the 95th percentile; the horizontal line indicates the median, and the error bars show the range of the data.
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itors, such as SOCS1 and SOCS3, were not expressed in significant amounts in these biopsy specimens (data not shown). Pretreatment levels of IRF1 and IRF2 mRNAs in wart biopsies. IRF1 mRNA levels were significantly higher in the complete responder group than in the incomplete responders, while IRF2 mRNA was expressed in significantly larger amounts in biopsy specimens from incomplete responders (Fig. 3A). Accordingly, the IRF1:IRF2 ratio was significantly higher in complete responders than in incomplete responders. Pretreatment levels of mRNAs of endogenous IFNs in wart biopsy specimens. IFN-␣ and IFN-␥ mRNA levels were determined similarly, as described above (Fig. 3B). Wart biopsy specimens from complete responders contained significantly higher levels of IFN-␣ and IFN-␥ mRNAs than did those of incomplete responders. Status of differentiation of biopsy specimens before treatment. mRNAs for markers of cellular differentiation such as involucrin and keratin 10, but not keratinocyte transglutaminase, were expressed at significantly higher levels in biopsy specimens from complete responders than in those of incomplete responders before treatment (Fig. 4). Effects of differentiation on constitutive expression of IFNresponsive genes as well as on IQ-induced expression of IFNinduced genes in normal human keratinocytes in vitro. mRNA levels of STATs, IRFs, and PIASs were determined in cultures of normal human keratinocytes (Fig. 5A) under differentiation-inducing and non-differentiation-inducing conditions. Differentiation was induced by adding calcium to the medium as described in Materials and Methods. Interestingly, constitutive expression of STAT1 and IRF1 was increased with differentiation. In contrast, STAT3 and PIAS1 mRNA levels decreased while levels of IRF2 and PIAS3 mRNAs were unchanged or moderately changed, respectively, upon differentiation. In addition, differentiation significantly increased the inducibility of 2⬘-5⬘-oligo(A) synthetase (OAS) and RNA-dependent protein kinase (PKR) mRNAs by IQ in cultures (Fig. 5B). DISCUSSION IQ induces IFN-␣ and several other cytokines (12, 23, 37) that are believed to be part of its mechanism of action (13, 33). IQ-induced IFN and cytokine expression as well as activation of IFN-stimulated genes requires STAT1 (6); thus, IQ may utilize the JAK/STAT pathway or STAT1 may be important for IFN-␣ induction (9, 34). Recent studies revealed that the duration and intensity of a cell’s response to cytokines appear to be determined by the net effect of several regulatory mechanisms. Therefore, the balance between positive and negative regulators of the JAK/STAT pathway may influence the effectiveness of IQ therapies. STAT proteins are effectors of cytokine and growth factor signaling (20, 21, 25). STATs are activated via tyrosine kinases (JAK kinases) and then transported to the nucleus, where they bind DNA response elements in promoters and regulate gene expression (34). STAT1 levels are significantly higher in complete responders to IQ than in incomplete responders (Fig. 2B). This is in agreement with the fact that STAT1 is essential for induction of various genes by IQ (6). In contrast, STAT3 levels were higher in incomplete responders (Fig. 2A). STAT3 is activated by growth factors, so it is possibly involved in mitogenic signaling (11). It is interesting that a STAT-binding sequence located in the c-myc P2 promoter (22) preferably binds STAT3 but not STAT1 and thus activates c-myc transcription. Also, the profile of activated STATs (i.e., the relative stoichiometry of various STAT family members) may be an important determinant of the biological outcome of signaling.
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FIG. 4. Analysis of status of differentiation in wart biopsy specimens. Biopsy specimens from complete responders and from incomplete responders were analyzed by a semiquantitative RT-PCR method. cDNAs were obtained from the isolated RNAs by RT and subjected to PCR with gene-specific primer pairs. PCR fragments were resolved on an agarose gel and transferred to a nylon membrane. Hybridization with gene-specific probes and subsequent autoradiography revealed the identities of fragments, and quantitation was done by densitometry. Data are expressed as ratios of target gene and G3PDH mRNA levels. Data were plotted as box plots and analyzed for statistical differences. The box extends from the 5th to the 95th percentile; the horizontal line indicates the median, and the error bars show the range of the data. N.S., not significant; INV, involucrin; K10, keratin 10; KTG, keratinocyte transglutaminase.
Heterodimers of STAT1 and STAT3 differ in their sequence preferences and transcriptional induction properties (24). In addition, preexisting, latent STAT complexes are more likely targeted by cytokine stimulation than are newly associated complexes (32). Thus, the pretreatment levels of various STATs may determine the overall responses to IQ. Preexisting levels of various cytokines induced a distinct combinatorial assembly of STATs (39). Since the constitutive levels of IFN-␣ and IFN-␥ in the two groups of subjects are significantly different (Fig. 3B), we assumed that they might determine levels of STATs through constitutive activation. While keratinocytes express IFN-␣ (27, 28), the source of IFN-␥ may be resident or infiltrating T cells (29). Wart biopsy specimens responding to IFN therapy contain higher constitutive levels of markers of T-cell infiltration than do specimens from incomplete responders (3, 4). Another possibility is that IQ responses are primed by combinations of IFN-␣ and IFN-␥ (M. Tomai, personal communication). In addition, differentiation supports constitutive expression of STAT1 but not STAT3 (Fig. 5A) and complete responders exhibit a more highly differentiated phenotype (Fig. 4). Thus, distinct cytokine levels and/or differentiation status may determine the outcome of cytokine responses. IRFs are a family of transcriptional regulators that exert distinct roles in biological processes such as pathogen responses, cytokine signaling, cell growth regulation, and hematopoietic development (31). Two well-studied members of this family, IRF1 and IRF2, have antagonistic roles in gene regulation: they bind to the same DNA elements, but IRF1 acts as a stimulator of transcription while IRF2 acts as a repressor
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FIG. 5. Impact of cellular differentiation on constitutive and IQ-induced mRNA expression of IFN-responsive genes in vitro. (A) Primary normal human keratinocytes were cultured under non-differentiation-inducing or differentiation-inducing conditions as described in Materials and Methods. Constitutive mRNA levels of STATs, IRFs, and PIASs were determined by RT-PCR. cDNAs were obtained from the isolated RNAs by RT and subjected to PCR with gene-specific primer pairs. PCR fragments were resolved on an agarose gel and transferred to a nylon membrane. Hybridization with gene-specific probes and subsequent autoradiography revealed the identities of fragments, and quantitation was done by densitometry. mRNA levels are expressed as ratios of mRNA levels of target gene and a constitutively expressed gene (encoding G3PDH). (B) Keratinocytes were treated with 5 g of IQ/ml for 72 h under non-differentiation-inducing or differentiation-inducing conditions. Induced levels of OAS and PKR were determined by RT-PCR. Results are shown as ratios of values for treated and untreated keratinocytes.
(14). Apparently, alterations of the IRF1:IRF2 ratio can have significant consequences for cell growth (36); restrained cell growth depends on a balance between these two mutually antagonistic transcription factors (16). During immune responses, IRF1 activation is necessary for major histocompatibility complex upregulation (18, 36). The IRF1:IRF2 ratio in biopsy specimens from complete responders is high (Fig. 3A). This is due to the fact that constitutive IRF1 levels are higher but IRF2 levels are lower in biopsy specimens from complete responders. These differences may reflect differences in the levels of STATs (Fig. 2A); STAT1 binds to and activates the IRF1 promoter (15). High IRF1:IRF2 ratios are also advantageous for good IFN responses in persons with myeloid leukemias (19). By analogy, we can assume that the higher IRF1:IRF2 ratio of complete responders is a predictor of better responses to IQ therapy. Cytokines induce a variety of biological responses by binding to specific cell surface receptors and activating cytoplasmic signal transduction pathways, such as the JAK/STAT pathway. Recent studies have identified two new families of negative regulatory molecules, SOCS and PIAS, which function in novel ways to suppress signal transduction pathways (35). Apparently, PIAS1, but not PIAS3, is expressed at higher levels in biopsy specimens of incomplete responders than in those of complete responders (Fig. 2B). PIAS1 blocks the DNA binding activity of STAT1 and inhibits STAT1-mediated gene activation in response to IFN (26). These results, together with the data on constitutive levels of STAT1 (Fig. 2A), are in
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agreement with the finding that STAT1 is essential for an optimum IFN response to IQ (6). Although the data presented above offer an explanation for differences in clinical responses to IQ treatment, they do not explain the cause of those differences. One possible explanation is the state of cellular differentiation in the patients who exhibit better clearance: complete responders are more differentiated than the incomplete responders (Fig. 4). Our previous study of genital warts also emphasized the role of differentiation in IFN responses (2). Differentiation influences IQ responses in two ways. First, differentiation affects constitutive expression of STATs and IRFs (Fig. 5A): STAT1 and IRF1 are positively influenced by differentiation, while STAT3 and PIAS1 are negatively correlated with higher-level differentiation. The latter observation for PIAS1 may account for its higher-level expression in incomplete responders (Fig. 2B) who are otherwise less differentiated (Fig. 4). Second, differentiation potentiates IQ-mediated activation of IFN response genes (Fig. 5B). In summary, we postulate that high constitutive levels of STAT1 and IRF1 but low levels of IRF2 and PIAS1 are essential for a complete response to IQ therapy. Constitutive mRNA levels of these genes are affected by cellular differentiation influencing IQ-induced gene expression. The cause of altered differentiation, however, remains to be determined. REFERENCES 1. Anonymous. 1997. Imiquimod for genital warts. Med. Lett. Drugs Ther. 39:118–119. 2. Arany, I., M. M. Brysk, H. Brysk, and S. K. Tyring. 1996. Response to interferon treatment decreases with epidermal dedifferentiation in condylomas. Antivir. Res. 32:19–26. 3. Arany, I., and S. K. Tyring. 1996. Activation of local cell-mediated immunity in interferon responsive patients with human papillomavirus-associated lesions. J. Interferon Cytokine Res. 16:453–460. 4. Arany, I., and S. K. Tyring. 1996. Status of local cellular immunity in interferon responsive and nonresponsive human papillomavirus-associated lesions. Sex. Transm. Dis. 26:475–480. 5. Beutner, K. R., S. L. Spruance, A. J. Hougham, T. L. Fox, M. L. Owens, and J. M. Douglas, Jr. 1998. Treatment of genital warts with an immune-response modifier (imiquimod). J. Am. Acad. Dermatol. 38:230–239. 6. Bottrel, R. L. A., Y.-L. Yang, D. E. Levy, M. Tomai, and L. F. L. Reis. 1999. The immune response modifier imiquimod requires STAT-1 for induction of interferon, interferon-stimulated genes, and interleukin-6. Antimicrob. Agents Chemother. 43:856–861. 7. Brysk, M. M., I. Arany, H. Brysk, S.-H. Chen, K. H. Calhoun, and S. K. Tyring. 1995. Epithelial cell responsiveness to interferon gamma: epidermis vs. buccal mucosa. Mol. Cell. Differ. 3:213–223. 8. Brysk, M. M., I. Arany, H. Brysk, S.-H. Chen, K. H. Calhoun, and S. K. Tyring. 1995. Gene expression of markers associated with proliferation and differentiation in keratinocytes cultured from skin and from oral mucosa. Arch. Oral Biol. 40:855–862. 9. Darnell, J. E. 1997. STATs and gene regulation. Science 277:1630–1635. 10. Edwards, L., A. Ferenczy, L. Eron, D. Baker, M. L. Owens, T. L. Fox, A. J. Hougham, and K. A. Schmitt. 1998. Self-administered topical 5% imiquimod cream for external anogenital warts. Arch. Dermatol. 134:25–30. 11. Garcia, R., and R. Jove. 1998. Activation of STAT transcription factors in oncogenic tyrosine kinase signaling. J. Biomed. Sci. 5:79–85. 12. Gibson, S. J., L. M. Imbertson, T. L. Wagner, T. L. Testerman, M. J. Reiter, R. L. Miller, and M. A. Tomai. 1995. Cellular requirements for cytokine production in response to the immunomodulators imiquimod and S-27609. J. Interferon Cytokine Res. 15:537–545. 13. Gross, G. 1997. Therapy of human papillomavirus infection and associated epithelial tumors. Intervirology 40:368–377. 14. Harada, H., T. Fujita, M. Miyamoto, Y. Kimura, M. Maruyama, A. Furia, T. Miyata, and T. Taniguchi. 1989. Structurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN and IFN-inducible genes. Cell 58:729–739. 15. Harada, H., E.-I. Takahashi, S. Itoh, K. Harada, T.-A. Hori, and T. Taniguchi. 1994. Structure and regulation of the human interferon regulatory factor 1 (IRF-1) and IRF-2 genes: implications for a gene network in the interferon system. Mol. Cell. Biol. 14:1500–1509. 16. Harada, H., T. Taniguchi, and N. Tanaka. 1998. The role of interferon regulatory factors in the interferon system and cell growth. Biochimie 80: 641–650. 17. Hennings, H., D. Michael, C. Cheng, P. Steinert, K. Holbrook, and S. H.
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