Production of chemokines by tumorigenic keratinocyte cell lines harboring human papillomavirus (HPV) type 16

Chemkines

23 June 1997 - Poster presentations

P.3.01.08

Production of chemokines by tumorlgenic keratlnocyte cell lines harboring human papillomavirus (HPV) type 16

Magdalena Malejczyk ‘, Tomasz Gtzela 2, Sfawomir Majewski ‘, Thomas Schwarz3. Stefania Jabforiska’, Jacek Matejczyk2. ‘Deparfmenf of Dermatology Warsaw, Poland, 2 Department of Histology and Embryotogy Institute ofBiostructum, Warsaw Medical School, Warsaw,Poland, 3Labomtory for Cell Biology, Ludwig Boltzmann Institute, Department of Demtatotogy University of Mfinster, Mgnster, Germany Intmductlon: The lesions induced by potentially oncogenic (“high risk”) HPV types usually regress. However, a small proportion of lesions persists and may undergo malignant conversion. The mechanisms responsible for regression or malignant progression of HPV-associated lesions are still poorly recognized. Regression is mediated by inflttrating lymphocytes and monocytes. Thus, recruitment of lymphoid cells to the lesion may be a crucial step in rejection of HPVtransformed keratinocytes. Accumulation of lymphoid cells at site of immunological reaction depends on local production of chemotactic factors, especially chemoklnes. Therefore, the aim of our present study was to evaluate expression and production of various chemokines by different keratinocyte/epithelial cell lines harboring and expressing HPV16 DNA sequences. Mat&al and Methods: The study was performed on tumotigenic HPVl6-harbortng SKv, SiHa, and CaSki cell lines and nontumorigenic HaCaT keratinccyte cell line which does not contain HPV. The presence of MCP-1, RANTES, and IL-8 in cell-free culture supematants was evaluated by specific ELISA and Western blot analysis. Expression of specific chemokine mRNAs was analyzed by transcription-pdymerase chain reaction and Northern blot analysis. Chemotactic activity for mononuclear blood cells and granulocytes in cell-condkioned media was tested using the Transwell” migration chambers (Costar, USA). Reauk Nontumorigenic HaCaT cells spontaneously released a relatively high amounts of MCP-1, RANTES, and IL-E. Stimulation of HaCaT cells with mTNFa resulted in significant increase of production of these chemokines. On the contrary, highly tumongenic CaSki cells released neither MCP-1 nor RANTES and production of these chemokines could not be upregulated by rhTNFa. Less tumorigenic SKv and SiHa cells released only small quanthies of MCP-1 and RANTES and their upregulation by rhTNF-o was relatively low. In all tested cell lines production of chemokines correlated with respective steady-state specific mRNA levels. Production of MCP-1 and RANTES by the tested cells also correlated with an ability of cell-conditioned media to stimulate chemotaxis of mononuclear blood cells. Interestingly. all HPV-harboring cell lines expressed and released high amounts of IL-8 and their culture supernatants displayed high chemotactic activity toward granulocytes. Conclusion: The results of our study show that, consistently with previous reports, keratinocytes are able to express and release various chemokines. However, C-C chemokine expression is abrogated in HPV-harboring cells. This abrogation appears to be associated with higher tumotigenic potential of HPVharboring cells and might be responsible for inability to reject HPV-associated malignant tumors. The exact mechanism of this abrogated C-C chemokine expression remains to be elucidated.

P.3.01.09

Differential regulatlon of RANTES production on prlmarv human T-cells followlna combinations of CDZi/Cb28/PMA treatments -

Y. Sotsios, S.G. Ward, J. Westwick. School ofPhannacyand fhamtacotogy University of Bath, Claverton Down, Bath, United Kingdom Introduction: RANTES is a member of the C-C chemokine family and has been implicated in the pathogenesis of allergic inflammatory conditions induding asthma and rhinitfs. Moreover, members of the C-C chemokine family have been shown to interfere with M-tropic HIV-l entry. Apart from its role in directional migration of T-cells, RANTES has also been proposed to provide costimulation, causing proliferation and lymphocyte activation. However, there is still some contradiction with the available results. In this study the role of RANTES during the initial activation of resting purified CD2+/CD3+ human T-cells and PHAAL-2 expanded T-lymphoblasts was addressed. hhterials and Methods: PBCs were obtained from healthy donors and Tcells were negatively selected so as to minimise activation during handling. The resulting CD4+ and CD8+ population was activated either through ligation of the TCWCDI complex and CD28, or with phorbol my&ate acetate alone and with CD28 costimulation. From the resting prknary T-cells, RANTES production kinetics over 96 hours following the above stimulatory conditions was determined with ELISA and compared to 3H-thymidine incorporation proliferative responses at 72 hours from identical stimulations. RANTES production was also measured from T-lymphoblasts over a dose range of hrll-2 (0.330 @ml) for up to 5 days after PHA stimulation. Results: At every time point examined, RANTES produced by primary CD3+ T cells from stimulation with PMA alone and together with CD28 was 2-7 fold greater compared to CD3/CD28 ligation. However, PMA alone did not cause

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dose-related augmentation of FtANTES production in contrast to CD3/CD28 and even PMA/CD28 stimulation. These results did not reflect the prolleratlve responses from the same conditions. PMA/CDPI and CD3/CD28 induced proliferation at the same levels whereas PMA alone induced very lie proliferation. CD3+ T-lymphoblasts produced basal levels of RANTES comparable to CD31CD28 stimulated prfmary T-cells. The addition of IL-2 (0.330 ns/ml) to the T-lymphoblasts did not increase the level of RANTES production during the 6-96 hour observation period compared to RANTES production in the absence of added IL-2. Concfuslon: To determine the contribution of RANTES to T-cell activation and epitope expression we are determining the time course of expression of the 3 chemokine receptors (CCR-1, CCR-4, CCR-5) and the effect of neutralising antibodies.

P.3.01 .lO

Cloning of human and murlne TERl, an orphan chemoklne receptor expressed by lymphold tissues, and CHRB a seven transmembrane spanning receptor

A. Zingoni ‘, G. Bemardini ‘, G. Spinetti ‘, A. Nista', C.T. Stortazzi 3, M. Rocchi 3, A. Santoni 1.2,M. Napolitano ‘. ‘Laboratory of Pathopbysiology, Regina Elena Cancer Institute, Rome, 2Department of Experimental Medicine and Pathology University of Rome, “La Sapienza’: /&r/y 3Genetics Institute, University of Bari, /m/y Introduction: A number of CC and CXC chemokine receptor genes has been cloned and shown to belong to a superfamily of seven transmembrane spanning receptors. We have attempted to isolate novel chemokine receptor genes and characterize them biochemically and functionally. Matertal and Methods: A PCR amplification with degenerate primers spanning conserved regions of chemokine receptors was canted out using the cDNA of K562 as template. The 200 bp probe obtained (70% homology to CCRI), named TERl, was used to screen the AFIX human genomic library. The isolated human complete ORF was labeled and hybridized to the mutine 129SV genomic library. Pdy-A+RNA from human tissues and cell lines were hybridized to the labeled TERI cDNA. A fluorescent “in situ” hybridization (FISH) of 10 Kb TERl and CHR3 genomic probes to human metaphases were used to map the corresponding genes. Results: We have isolated human and murine genomic clones of TERl, a novel orphan chemokine receptor gene expressed by thymus, spleen and, at low level, by PBL. The lymphoid cell lines MOLT-4, Hut78 and NK3.3 express abundant levels of the 4 Kb TERl transcript. The TERI ORF encodes a protein of 355 aminoacids that shows 40-43% homology with CC chemokine receptors. The TERl and CHR3 genes map on chromosome 3~21 and 6q27, respectively. Conclusion: TERl seems to represent a novel chernokine receptor gene whose expression is restricted to lymphoid tissues and cell lines. The molecular cloning of such receptor will allow to define ligand binding patterns and to analyse its role in thymic physiology. The CHR3 gene may belong to a novel seven transmembrane spanning receptor subfamily.

P.3.01 .ll

Chemokine receptors In granuloma formatlon during mycobacterial Infection

C. Bronke’, D.A. Smith’, T.N.C. Wells2, C.A. Power2, A.J. Hoogewerf2, S. Ehlers 3, G.J. Bancroft ’ ’ London School of Hygiene 81TropicalMedicine, Department of Clinical Sciences, Keppet Street, London, UK, 2Glaxo institute for Molecular Biology 14 Chemin des Au/x, 1228 Plan-/es-Ouates, Geneva, Switzerland, 3Forschungszentrum Bomtel, Division of Molecular Biology Borstel, Germany Introduction: Characteristics of the host response to mycobactedal infection are phagocyte and lymphocyte recruitment to sites of infection and granuloma formation. Initial studies using M. avium to infect immunocompetent versus immunodeficient mice have shown that granuloma formation and cell recruitment occurs in a T cell independent manner through IFNy secretion by NK cells. Macrophages infected with mycobacterta secrete TNFu and IL-12 which synergistically can activate NK cells. The presence of T lymphocytes, however, induces a more rapid and more focussed response. Our studies focus on the mechanisms of cell recruitment and the formation of granulomas in the absence of bacterial resistance. Materials & Methods: SCID mice were infected intravenously with a virulent strain of M. avium to study mechanisms of T cell independent granuloma formation. Mice then were treated with neutralising antibodies to either IFNy, TNFa or depleted of NK cells. Liver, lung and spleen samples were analyssd by histology, RT-PCR, CFU count. In situ hybridisation and cell type colocalisaton studies of histology sections were performed using RNA probes for chemokine receptors. Resuftm In viva infection with M. avium led to granuloma formation in SCID mice which could be ablated with either anti-TNFa or anti-IFNy treatment. Anal-