Patterns of cytokine expression in AIDS-related non-Hodgkin's lymphoma
Dr Giuseppe Saglio Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Medicina Interna 5A, Ospedale San Luigi Gonzaga, Regione Gonzole 10, 10043 Orbassano-Torino, Italy.
The pathogenesis of AIDS-related non-Hodgkin's lymphomas (AIDS-NHL) involves accumulation of genetic lesions, stimulation and selection by antigen, as well as infection by viruses. Deregulation of cytokine loops has also been proposed to contribute to AIDS-NHL development, although data are available only for a limited number of cytokines. In this study we have utilized a panel of AIDS-NHL cell lines to investigate in detail the pattern of tumour expression and production of a wide spectrum of cytokines. The cytokines investigated included interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, TNFα, TNFβ, IFNγ, TGFβ2, G-CSF, GM-CSF and SCF. The AIDS-NHL cell lines utilized were representative of both AIDS-related Burkitt lymphoma (AIDS-BL) and AIDS-related body cavity-based lymphoma (AIDS-BCBL). Overall, AIDS-NHL were found to produce IL-6, IL-10 and TNFβ, although with different patterns depending upon the biological features of the tumour. Production of high levels of IL10 preferentially associated with Epstein-Barr virus (EBV) positive AIDS-BL and AIDS-BCBL, although lower levels of the cytokine were also detectable among EBV-negative AIDS-BL. Production of IL-6 was restricted to EBV-positive AIDS-BL and AIDS-BCBL, whereas it was absent among EBV-negative AIDS-BL. Production of TNFβ clustered with AIDS-BL, whereas this was absent among AIDS-BCBL. These results define that the pattern of cytokine expression of AIDS-NHL depends upon the biological features of the tumour and may have implications for the pathogenesis of these disorders.
Non-Hodgkin's lymphomas (NHL) represent the second most frequent neoplasm associated with AIDS, and their frequency has been increasing steadily since the beginning of the AIDS epidemic ( Beral et al, 1991 ; Karp & Broder, 1991; Levine, 1992). AIDS-related NHL (AIDS-NHL) are consistently of B-cell origin and belong to distinct pathologic categories, including Burkitt's lymphoma (BL), diffuse large cell lymphoma (DLCL) and body-cavity-based lymphoma (BCBL) ( Beral et al, 1991 ; Karp & Broder, 1991; Levine, 1992; Cesarman et al, 1995 a; Gaidano & Carbone, 1995; Green et al, 1995 ). The molecular pathogenesis of AIDS-NHL has been clarified to a certain extent, leading to the concept that distinct histologic types of AIDS-NHL associate with different molecular pathways ( Gaidano & Carbone, 1995; Gaidano & Dalla-Favera, 1995). Thus, AIDS-BL are characterized by c-myc rearrangements, p53 inactivation and Epstein-Barr virus (EBV) infection in 100%, 60% and 30% of cases respectively. AIDS-DLCL associate with EBV infection in the large majority of cases and with bcl-6 rearrangements in a fraction of cases. Finally, infection by human herpesvirus type 8 (HHV-8) is associated with 100% AIDS-BCBL, which frequently also carry the EBV genome.
In addition to genetic alterations and viral infection, the pathogenesis of AIDS-NHL is thought to involve also deregulation of cytokine loops. This notion stems from the observation that HIV infection deregulates networks of several cytokines which are known to be implicated in the proliferation and differentiation of B cells ( Amadori & Chieco-Bianchi, 1990; Fauci et al, 1991 ; Moses et al, 1998 ). For example, it is well established that HIV may induce in vivo expansion of activated B cells secreting IL-6, which may potentially acquire growth advantage and become more prone to transformation ( Amadori & Chieco-Bianchi, 1990; Rieckmann et al, 1991 ; Boue et al, 1992 ; Moses et al, 1998 ). Similarly, TNFβ, which may act as a growth factor for B-cell neoplasms, is induced by HIV in B cells ( Sastry et al, 1990 ; Gibbons et al, 1994 ). Although a number of studies have concentrated on the involvement of selected cytokines in AIDS-BL pathogenesis ( Benjamin et al, 1992 ; Emilie et al, 1992a , b; Masoodet al, 1995 ), a comprehensive analysis of cytokine expression in different AIDS-NHL histologic categories is lacking and represented the aim of this study.
In the present study we have investigated the pattern of expression and production of multiple cytokines in AIDS-BL and AIDS-BCBL and we have compared it to the cytokine pattern of NHL of similar histology derived from the immunocompetent host. We have observed that AIDS-BL produce and release interleukin (IL)-10 and tumour necrosis factor (TNF)β in a significant proportion of cases and IL-6 in a fraction of samples. Moreover, our characterization of cytokines in AIDS-BCBL indicates that this new type of lymphoma associates with production of IL-6 and high levels of IL-10.
MATERIALS AND METHODS
Cell lines and cell culture
This study included a panel of 15 AIDS-NHL cell lines, representative of AIDS-BL and AIDS-BCBL, as well as a panel of 16 cell lines of AIDS-unrelated BL (sporadic and endemic BL). All cell lines investigated in this study have been previously characterized in detail ( Lenoir et al, 1982 ; Ganser et al, 1988 ; Kiwanuka et al, 1988 ; Gaidano et al, 1993 , 1996; Roncella et al, 1993 ; Jain et al, 1994 ; Cesarman et al, 1995b ). AIDS-BL cell lines included HBL-1, HBL-2, HBL-3, HBL-4, PA682, As283A, ES III, LAMC3+, BRG IgM, BRG IgA, LAMC4+ and LAMG10. AIDS-BCBL cell lines included HBL-6, BC-1 and BC-2. Cell lines representative of sporadic BL (sBL) included Ramos, MC116, JD38, CA46, ST486, BL30, BL41, BL46, BL64 and BL108. Cell lines representative of endemic BL (eBL) included EB3, P3HR1, Daudi, Raji, Namalwa and BL135.
Cell lines were grown in suspension culture in RPMI 1640 containing 10% Fetal Bovine Serum (Hyclone Laboratories Inc., Logan, Utah), 2 m M L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomicin, at 37°C in 5% CO2 humidified atmosphere.
All the oligonucleotides used as primers for the polymerase chain reaction (PCR) were synthesized using an Applied Biosystems (Foster City, Calif.) synthesizer. Names and sequences of primers used for the following cytokines have been previously reported: IL-1α, IL-1β, IL-2, IL-4, G-CSF ( Sorg et al, 1991 ); IL-5, IL-6, IL-7, TGFβ2 ( Filgueira et al, 1993 ); IL-8, IL-10, TNFα, IFNγ, GM-CSF ( Lagoo-Deenadayalan et al, 1993 ); TNFβ ( Villinger et al, 1993 ). The primers chosen for IL-6 amplification were specific for the cellular IL-6 gene, as opposed to the HHV-8 encoded IL-6 ( Moore et al, 1996 ). PCR primer sequences for IL-3 ( Yang et al, 1986 ), SCF ( Martin et al, 1990 ), β actin ( Nakajima et al, 1985 ) and IL-13 ( MacKenzie et al, 1993 ) were as follows: IL3-5′ 5′-ATGAGCCGCCTGCCCGTCCTG-3′ and IL3-3′ 5′-GCGAGGCTCAAAGTCGTCTGTTG-3′ (for IL3); SCF-5′ 5′-CCATTGATGCCTTCAAGGAC-3′ and SCF-3′ 5′-GGCTGTCTCTTCTTCCAGTA-3′ (for SCF); β actin-5′ 5′-ACACTGTGCCCATCTACGAGGGG-3′, and β actin-3′ 5′-ATGATGGAGTTGAAGGTAGTTTCGTGGAT-3′ (for β actin); IL13-5′ 5′-GACCACGGTCATTGCTCTCACTTGC-3′, and IL13-3′ 5′-TCGATTTTGGTGTCTCGGACATG-3′ (for IL-13).
RNA extraction and RT-PCR
Total cytoplasmic RNA was extracted from 10–20 × 106cells using a commercially available kit (Ultraspec RNA Isolation System, Biotecx Laboratories Inc., Texas). The purity and integrity of each RNA sample was checked on a formamide-agarose gel ( Sambrook et al, 1989 ). RT-PCR reactions were performed by using the GeneAmp RNA PCR Kit (Perkin Elmer, Roche Molecular Systems Inc., N.J.), according to the manufacturer's instructions. Briefly, cDNA was synthesized from 1 μg of total RNA in Perkin Elmer PCR buffer II containing 5 m M MgCl2, 1 m M each dNTP, 1 U/μl RNase inibitor, 2.5 U/μl MuLV Reverse Transcriptase, 2.5 μM Oligo d(T)16 primer, in a final volume of 20 μl, at 42°C for 45 min. PCR reactions were then performed as follows: 10 μl of RT reaction product, Perkin Elmer PCR buffer II, 1.5 m M MgCl2, 0.15 μM of each primer, 2.5 U AmpliTaq DNA Polymerase, in a final volume of 50 μl. 35 cycles of denaturation (94°C), annealing (annealing temperature was optimized for each pair of primers) and extension (72°C) were performed in a Hybaid Omn-E Thermal Cycler. In order to eliminate spurious amplification in the PCR reaction, the amplification of IL-6, TNFβ and TGFβ2 was performed according to the Touchdown protocol as described by Don et al (1991 ). The PCR products were then visualized by ethidium bromide staining on a 2% agarose gel. Specificity of each PCR product was established by correspondence to the expected size and by restriction analysis with at least three different restriction enzymes. All RT-PCR experiments included appropriate positive and negative controls which were represented by cell lines or peripheral blood mononuclear cells known to express (or not express) a given cytokine.
Immunodetection of cytokines in cell line supernatants was performed for IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-13, TNFα, TNFβ and IFNγ. To collect supernatants, all cell lines were grown from 5 × 105 to 2 × 106 cells/ml. After cell sedimentation by centrifugation, the medium was filtered with a 0.22 μm filter, aliquoted and stored at −80°C until use. The supernatants were tested for the concentration of IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, TNFα and IFNγ by a sandwich ELISA method ( Elsasser-Beila et al, 1991 ). Antibodies against IL-1β, IL-2, IL-4, IL-6, TNFα and IFNγ were provided by Hoffmann-la Roche, Basel, Switzerland. Antibodies against IL-7 and IL-10 were purchased from Pharmingen, San Diego, Calif. TNFβ (R&D Systems Inc., Minneapolis, Min.) and IL-13 (Chemicon International Inc., Temecula, Calif.) were quantified by specific ELISA assays (commercial), according to the manufacturer's recommendations.
In the present study the following cytokines were investigated for expression and/or production in AIDS-NHL and, for comparison, in AIDS-unrelated BL: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, TNFα, TNFβ, IFNγ, TGFβ2, G-CSF, GM-CSF and SCF. Analysis of cytokine expression and production was performed by a two-step strategy. As a first step, all cytokines were tested for expression at the RNA level by RT-PCR in all samples included in the study. Subsequently, cytokines scored positive by the RT-PCR assay in one or more lymphoma cell lines were further investigated by specific ELISA assays, in order to assess the production and secretion of the cytokine protein.
Overall, the following cytokines were scored positive for protein production and secretion by AIDS-NHL: IL-6, IL-10 and TNFβ ( Table I and Fig 1). All other cytokines tested in AIDS-NHL scored either negative in RT-PCR experiments or displayed a sporadic positivity by RT-PCR in the absence of protein production and secretion. Conceivably, the absence of protein production in cell lines that were positive by RT-PCR could be ascribed to either very low levels of cytokine transcripts, which were insufficient to yield detectable amounts of cytokine molecules or, alternatively, to post-transcriptional regulation of cytokine expression. Cytokines which were expressed at both transcript and protein level, i.e. IL-6, IL-10 and TNFβ, displayed a different pattern of association with distinct biological categories of AIDS-NHL.
Overall, cases of AIDS-BL were positive for the production and secretion of IL-6, IL-10 and TNFβ, although in different proportions and at different levels ( Tables I and II). Production of IL-10 occurred in the majority of AIDS-BL tested (9/12), including 7/7 EBV-positive and 2/5 EBV-negative cases. Notably, among AIDS-BL cases producing IL-10 the cytokine was generally secreted at substantially higher levels in cases harbouring EBV infection than in cases devoid of the virus ( Table II). In particular, two cases of EBV-positive AIDS-BL (HBL-4 and PA682) secreted levels of IL-10 which were 10–100-fold higher than levels secreted by other AIDS-BL cell lines. The rate of IL-10 production in AIDS-BL (9/12) as opposed to that of sporadic BL (3/10) and endemic BL (2/6) suggested that the cytokine was preferentially produced by AIDS-related cases. Also, the levels of IL-10 were generally higher among AIDS-BL, since 6/12 AIDS-BL, but only 1/16 AIDS-unrelated BL, produced >10 ng/ml IL-10.
In addition to IL-10, AIDS-BL also produced and secreted TNFβ (6/12) and, in a minority of cases, IL-6 (3/12). Notably, production of IL-6 was consistently negative among AIDS-unrelated BL (n = 16), whereas the frequency of TNFβ positivity was identical between AIDS-BL (6/12) and AIDS-unrelated BL (8/16).
When considering individual cases of AIDS-BL, the following patterns of cytokine production were observed. Two cases (HBL-1 and As283A) produced IL-10, TNFβ and IL-6, three cases (HBL-3, PA682, BRG IgM) produced IL-10 and TNFβ, one case (HBL-4) produced IL-6 and IL-10, whereas four cases produced only one cytokine, namely IL-10 in the instance of LAMC3+, LAMC4+ and LAMG10, and TNFβ in the instance of ES III.
All AIDS-BCBL were found to produce and secrete high levels of IL-6 and IL-10, whereas production of TNFβ was negative in all AIDS-BCBL tested ( Tables I and II). All cases of AIDS-BCBL secreted IL-6 and IL-10 at levels which were above the average levels produced by those of AIDS-BL.
The mechanisms leading to AIDS-associated lymphomas are not fully understood, but it appears that they may involve the disfunction of cytokine loops ( Fauci et al, 1991 ; Benjamin et al, 1992 ; Emilie et al, 1992a , b; Gaidano & Carbone, 1995 ; Gaidano & Dalla-Favera, 1995; Masood et al, 1995 ). Cytokines are a family of pleotropic regulatory molecules secreted by several cell types and involved in the regulation of haemopoietic and immune systems. In situations of immune suppression, as in the case of AIDS, disruption of the normal regulation of cytokine networks may lead to altered immune responses and contribute to the establishment of malignant outgrowths ( Amadori & Chieco-Bianchi, 1990). Multiple cytokines have been postulated to be important in the development of B-cell NHL, including molecules which physiologically regulate B-cell proliferation and differentiation and which may contribute to B-cell lymphomagenesis through autocrine or paracrine loops.
In this report we analysed the expression and production of 18 different cytokines in AIDS-BL and AIDS-BCBL cell lines. The aim of the study was twofold. Firstly, to define the cytokine profile of AIDS-NHL with respect to histology and viral infection. Secondly, in the case of AIDS-BL, to compare the cytokine profile of AIDS-related cases with that of AIDS-unrelated cases. Overall, out of the 18 molecules tested, only three cytokines were produced by AIDS-NHL, including IL-10, IL-6 and TNFβ. IL-10 was preferentially produced by EBV-positive AIDS-NHL, compared to all other tumours tested. Production of IL-6 was associated with AIDS-BCBL and with a fraction of AIDS-BL, whereas it was consistently absent in AIDS-unrelated BL. TNFβ was produced by both AIDS-related and AIDS-unrelated BL, but not by AIDS-BCBL.
IL-10 and IL-6 are key regulators of B-cell proliferation and differentiation. Patients with lymphoma and HIV infection have increased serum levels of IL-10 ( Emilie et al, 1992b ), which has been shown to be produced by AIDS-NHL cells, particularly if associated with EBV infection ( Benjamin et al, 1992 ; Masood et al, 1995 ). IL-10 appears to be pathogenetically relevant, since antisense oligonucleotides inhibited the in vitroproliferation of AIDS-NHL cells, suggesting an autocrine mechanism of growth ( Masood et al, 1995 ). Our data confirm and expand the spectrum of AIDS-NHL associated with IL-10 production. With respect to AIDS-BL, we show that production of IL-10 is not restricted to EBV-infected cases, although the cytokine levels in EBV-positive AIDS-BL are substantially higher than those found in EBV-negative AIDS-BL ( Benjamin et al, 1992 ; Emilie et al, 1992b ; this report). Curiously, when considering all BL variants carrying EBV infection, production of IL-10 appeared to preferentially associate with AIDS-related cases, since sporadic BL, which carries EBV infection in 30% of cases, as well as endemic BL, which consistently carry EBV infection, release IL-10 only in a minority of cases. The reasons for this discrepancy between AIDS-BL and AIDS-unrelated BL are presently unclear. In addition to AIDS-BL, our data demonstrated that AIDS-BCBL cell lines also produced and released large amounts of IL-10. Since AIDS-BCBL cell lines carried double infection by HHV-8 and EBV, it remains to be defined whether IL-10 production in AIDS-BCBL is related to the expression of viral antigens up-regulating IL-10 ( Nakagomi et al, 1994 ), or whether it reflects the differentiation stage or other biological properties of this lymphoma.
Several lines of evidence have linked IL-6 to lymphomagenesis. Serum level of IL-6 has been correlated with an increased risk of subsequent lymphoma in patients with AIDS ( Pluda et al, 1993 ). Furthermore, transgenic mice for IL-6 have been associated with the development of lymphoma ( Woodroofe et al, 1992 ). These observations have suggested that anti-IL6 antibodies may be used as therapeutic tools for AIDS-NHL ( Emilie et al, 1994 ). In the case of AIDS-related diffuse large cell lymphomas, IL-6 is thought to act as a paracrine growth factor, produced by accessory cells ( Emilie et al, 1992a ). Conversely, this and previous studies indicated that IL-6 was directly produced by a fraction of AIDS-BL and by most, if not all, AIDS-BCBL ( Benjamin et al, 1992 ; Moore et al, 1996 ; Chang, 1998). Among BL variants, production of IL-6 was restricted to EBV-positive cases of AIDS-BL, whereas it was consistently absent in all other BL variants, including EBV-negative AIDS-BL, all sporadic BL and all endemic BL. With respect to HHV-8-positive AIDS-BCBL, it should be noted that HHV-8 carries a gene encoding for viral IL-6, which can induce production of the cellular IL-6 ( Moore et al, 1996 ). Since our RT-PCR studies have defined that AIDS-BCBL specifically expressed the cellular IL-6 gene, it is conceivable that the measured levels of IL-6 protein were constituted by cellular IL-6, at least in part.
TNFβ, a member of the TNF family of ligands, induces B-cell proliferation and is essential for normal lymphoid development ( Matsumoto et al, 1996 ). Some NHL produce high levels of TNFβ which correlate with the presence of B symptoms ( Gibbons et al, 1994 ; Grüss & Dower, 1995). The pathogenetic relevance of TNFβ in lymphomagenesis was suggested by the observation that some AIDS-unrelated BL cell lines use TNFβ as an autocrine growth factor ( Gibbons et al, 1994 ). Our work shows that, in addition to AIDS-unrelated BL, a significant fraction of AIDS-related BL cell lines also produce TNFβ. Conversely, AIDS-BCBL cell lines were consistently negative for TNFβ production. Further investigations are needed to understand the precise contribution of TNFβ in the development of AIDS-BL, although, based on the example of AIDS-unrelated BL, it is conceivable that it may play a pathogenetic role also in cases associated with HIV infection ( Grüss & Dower, 1995).
Apart from directly stimulating lymphoma growth and survival, cytokines produced by AIDS-NHL may also affect the interplay between HIV, T cells and neoplastic B cells. In particular, IL-6 can up-regulate HIV production by infected cells and may cause T-cell proliferation, thereby expanding the pool of HIV-infected cells ( Rosenberg & Fauci, 1990; Doweiko & Groopman, 1998). On the other hand, the finding that AIDS-NHL produce IL-6 may be linked to the observation that HIV infection frequently associates with in vivo expansion of activated B cells secreting IL-6, which may acquire growth advantage and therefore become more prone to transformation ( Amadori & Chieco-Bianchi, 1990; Rieckmann et al, 1991 ; Boue et al, 1992 ; Moses et al, 1998 ). A similar phenomenon may be also postulated in the case of TNFβ, which was induced by HIV in B cells and may act as a growth factor for B-cell neoplasms ( Sastry et al, 1990 ; Gibbons et al, 1994 ). Regarding IL-10, the cytokine may not only act as an autocrine growth factor for the lymphoma ( Masood et al, 1995 ), but may also modulate the host immune response against the tumour ( Matsuda et al, 1994 ; Hagenbaugh et al, 1997 ; Zeidler et al, 1997 ). Also, IL-10 may increase HIV infection by activating the virus from latently and acutely infected monocytic cells and by increasing CCR5 expression in monocytes ( Finnegan et al, 1996 ; Sozzani et al, 1998 ).
Future studies are needed in order to confirm these data derived from AIDS-NHL cell lines and in the in vivo setting. Several observations, however, appear to validate the use of AIDS-NHL cell lines as an experimental model for the pathogenesis of these disorders. For example, in the case of IL-10, the expression profile of the cytokine and its relationship to tumour infection by EBV are similar in AIDS-NHL cell lines and in AIDS-NHL primary biopsies (this study; Benjamin et al, 1992 ; Emilie et al, 1992b ). In addition, investigations of AIDS-NHL pathogenetic features other than cytokine deregulation have confirmed that AIDS-NHL cell lines recapitulate the main biological features of these diseases, including the pattern of genetic alterations and viral infection ( Gaidano et al, 1998a , b).
The authors are indebted to Dr Ganser for the gift of the ES III cell line.
This work has been supported by ISS, Programma nazionale di ricerca sull'AIDS 1997–Progetto patologia clinica e terapia dell'AIDS (no. 30A.0.62 and 30A.0.10); by Fondazione Piera, Pietro e Giovanni Ferrero, Alba, Italy, and by Fondazione CRT, Torino, Italy. C.P. has been supported by a fellowship from A.I.R.C. P.G. is supported by a fellowship from the American–Italian Cancer Foundation. D.C. is supported by a fellowship from Fondazione Piera, Pietro e Giovanni Ferrero.