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Toll-like receptors 2, 4 and 9 expression in cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome)

European Journal of Dermatology. Volume 16, Number 6, 636-41, November-December 2006, Investigative report

DOI : 10.1684/ejd.2006.0088

Summary

Author(s) : V Jarrousse, G Quereux, S Marques-Briand, A-C Knol, A Khammari, B Dreno , INSERM U601, 9 quai Moncousu 44093 Nantes cedex 01, France, Clinique dermatologique, CHU Hotel-Dieu, 1 place A.Ricordeau, 44000 Nantes, France.

Summary : The aim of this work was to study Toll-like receptors (TLRs) 2, 4 and 9 expression patterns in parapsoriasis and in cutaneous T-cell lymphoma (CTCL): Mycosis fungoides (MF) and Sézary syndrome (SS) at different stages of the illness. The expression of TLRs was examined by immunohistochemistry on paraffin-embedded biopsies. Normal skin, atopic dermatitis and psoriasis, were used as controls. In cutaneous lesions of inflammatory diseases (atopic dermatitis, psoriasis) the expression of TLR2, TLR4 and TLR9 was low compared to normal skin. In parapsoriasis the expression of the three TLRs was similar to control. By contrast, in MF skin we observed a strong intensity of labelling with the three TLRs in the epidermis. Concerning SS, the expression of TLR2, TLR4 and TLR9 was intermediate between inflammatory lesions and MF. Thus, the development of skin lesions in MF appears associated with an increase of TLR2, TLR4 and TLR9 expression by keratinocytes in cutaneous lesions, which could play a role in the chronic activation of T lymphocytes in the skin.

Keywords : innate immunity, mycosis fungoides, Sézary syndrome, toll-like receptors

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ARTICLE

Auteur(s) : V Jarrousse¹, G Quereux², S Marques-Briand², A-C Knol¹, A Khammari^1,2, B Dreno^1,2

¹INSERM U601, 9 quai Moncousu 44093 Nantes cedex 01, France
²Clinique dermatologique, CHU Hotel-Dieu, 1 place A.Ricordeau, 44000 Nantes, France

accepté le 3 Août 2006

Toll-like receptors (TLRs) have been established to play an important role in activation of the innate immune response by recognizing various microbial-derived molecules. Currently the Human TLR family consists of 10 members (TLR1-TLR10) [1]. Recently a new member of the mammalian TLR family, TLR11, was described [2]. Each TLR is a single-pass transmembrane receptor with an extracellular domain containing multiple leucine rich repeats and an intracellular signalling domain that is homologous to the cytoplasmic tail of the IL1 receptor (TIR domain). Activation of TLRs induces strong inflammation and triggers anti-microbial responses and cytokine production via the nuclear factor-κB (NF-κB) signal transduction pathway. Different TLRs engage different combinations of adaptators: TLR1, TLR2, TLR4, TLR5, TLR7 and TLR9 trigger the MyD 88 dependent pathway and involve the early phase of NF-κB activation, which leads to the production of inflammatory cytokines [3]. TLR4 and TLR3 trigger the MyD 88 independent pathway and activate interferon (IFN)-regulatory factor (IRF-3) and involve the late phase of NF-κB activation, both of them leading to the production of INFβ and the expression of INF-inductible genes [4, 5]. Song et al. [6] have demonstrated that human keratinocytes expressed functional TLR4 and CD14. Another group [7] has shown that cultured keratinocytes expressed TLR2, TLR4 and MyD88, contrary to Kaway et al. [8] who could not detect TLR4 mRNA or protein expression in cultured keratinocytes.Epidermotropic Cutaneous T-cell Lymphoma (CTCL) is a lymphoproliferative disorder characterized by the clonal expansion of malignant CD4+ T-cells in the skin. They are represented by mycosis fungoides (MF) and Sézary syndrome (SS). MF is the most common manifestation of primary CTCL. The aetiology and exact steps in the pathogenesis of MF are not well understood. Most patients with MF first present with long-standing reactive inflammatory conditions such as parapsoriasis en plaques. Large plaque parapsoriasis is considered as an in situ stage of epidermotropic CTCL [9-11]. MF is distinguished from LPP by clinical criteria, histological criteria (infiltrate) and biological criteria (clonal TCR gene rearrangement) [12]. SS is considered as an erythrodermic leukemic variant of MF. Patients present with generalized erythroderma, lymphadenopathy, and circulating atypical T cells (Sézary cells) in the peripheral blood. The origin of this pathology remains unknown but the implication of viruses is highly suspected. Several papers have raised the hypothesis that a virus could play a role in the development of MF and SS. The main viruses were HTLV I [13, 14] and Epstein-Barr virus (EBV) but these hypotheses remain to be confirmed. Clinical observation (CTCL patients suffer often from Herpes infection) and histological observation (chronic lymphocytes T activation in epidermis is a feature of CTCL patients) [15], suggested to us that activation of Toll-like receptors by virus on keratinocytes could play a role in the development of the T lymphocytes infiltrate. Interestingly, a link has been demonstrated between TLR and infectious agents.TLR2 is involved in the response to a variety of bacterial components. They include peptidoglycan, lipoproteins, lipopeptides, and zymosan. These TLR2 ligands are probably recognized by a heterodimer formed between TLR2 and TLR6 or TLR1. TLR4 recognizes lipopolysaccharide (LPS), an integral component of the outer membrane of Gram-negative bacteria and a causative agent of endotoxin shock. Recognition of LPS requires not only TLR4, but also a TLR4 accessory molecule called MD2. TLR2 and TLR4 are expressed on the cell surface. TLR9 recognizes unmethylated 2’deoxyribo(cytidine-phosphate-guanosine) (CpG) DNA motifs that are more commonly found in bacterial and viral genomes and not in vertebrate genomes. TLR9 is localized in the endosomal/ vacuolar/ vesicular compartment, but not at the cell surface. At least Toll-like receptors appear to recognize viral infection with TLR4 (respiratory syncytial virus (RSV) and retrovirus infection), TLR2 (Herpesviridae) and TLR9 (Herpes viridae) [16].The aim of this work was to look for a new way of T cell activation in epidermotropic CTCL, thus, in this study, we investigated the expression of TLR2, TLR4 and TLR9 by keratinocytes in parapsoriasis, MF and SS.

Materials and methods

Patient samples

This study was performed on paraffin-embedded skin biopsies of plaque parapsoriasis (n = 6), early MF stage I (n = 12) according the criteria which have been recently defined [17], plaque MF (MF stage IIb) (n = 6) and SS (n = 19). For all these patients, the study was performed before treatment (T0). Five biopsies of psoriasis, five biopsies of atopic dermatitis, and five biopsies of healthy skin were used as controls.

Immunohistochemistry on cutaneous sections

Cutaneous sections prepared from paraffin-embedded biopsies were cut into 5μm sections and mounted on glass slides. The slides were deparaffinized and incubated for 30 min with Tris-buffered saline (TBS), Tween20 W/v (Sigma, St Louis, USA), bovine serum albumin (BSA) 0.1% (Sigma, St Louis, USA). They were then incubated for 30 min with the anti-TLR2 H-175, or the anti-TLR4 H-80, or anti-TLR9 D-18 polyclonal antibody (TEBU, Le Perray-en-Yvelines, France) at a concentration of 4 μg/mL. One slide was incubated without primary antibody and constituted a negative control for TLR2 and TLR4. To block TLR9 antibody, a 5-fold concentration of the corresponding specific peptide was incubated overnight at 4°C. The antibody was diluted to 4μg/ml. All slides were washed and incubated for 30 min with a secondary biotinylated antibody (ChemTek detection kit peroxidase/AEC, rabbit/Mouse, DAKO, Glostrup, Denmark), and then washed and further incubated for 30 min with streptavidin/peroxydase (DAKO, Glostrup, Denmark). After washing and incubation with biotinyl tyramide (indirect TSA kit, Perkin Elmer, Boston, USA) diluted 1/80 for 5 min, slides were washed again and incubated for a second time with streptavidin/peroxydase. After a final wash, reaction products were revealed using 3-amino-9-ethylcarbazole (AEC) (DAKO, Glostrup, Denmark) peroxidase substrate for 5 min. The reaction was stopped with distilled water (10 min) and counter-staining done with Mayer haemalum (VWR international, Fontenay-sous-Bois, France) for about 1 min. The positivity of the staining was quantified according to the intensity of labelling as none (–), weak (+), moderate (++) and strong (+++). Two different examiners viewed the slides.

Double immunostaining

Staining was carried out on serial cryostat sections which were fixed in acetone for 10 min at 4°C. The slides were incubated 15 min with phosphate buffered saline (PBS), bovine serum albumin (BSA) 0.1% (Sigma, St Louis, USA). They were then incubated for 30 min with the first primary antibody anti-TLR2 H-175 polyclonal antibody (TEBU, La peray-en-Yvelines, France) diluted at 1/3. Slides were then washed and incubated for 30 min with goat F(ab’) Fragment anti-rabbit IgG(H+L)-FITC (Beckman Coulter, Marseille, France) diluted at 1/10 and then washed and further incubated with goat normal serum diluted at 1/10 for 30 min. After washing and incubation with second primary antibody, CD3 or CD4 (DAKO, Trappes, France) diluted at 1/5 or CD1a (Beckman Coulter, Marseille, France) ready to use. Slides were washed and incubated 30 min with goat F(ab’)₂ Fragment anti-mouse IgG (H+L)-Biotin (Beckman Coulter, Marseille, France) diluted at 1/40, then washed and incubated with Streptavidine-PE-texas Red (Sav-PE-TxR) Conjugate (BD Biosciences, Le Pont De Claix, France). After a final wash, slides were mounted in GelTol Aqueous Mounting Medium (Immunotech, Marseille, France) and observed under a Leitz ARISTOPLAN microscope with an excitation wavelength 450-470 nm. Photographs were taken with a digital SLR camera D70S with an exposure of 8 seconds.

Statistics

The statistical significance of the data was determined by Chi 2 test. A P < 0.05 was taken as significant.

Results

Control samples

TLR2, TLR4 and TLR9 expression in normal skin (figures 1-3)

By immunohistochemistry on sections obtained from formalin-fixed and paraffin embedded normal human skin biopsies, we found that TLR2, TLR4 and TLR9 were weakly (+) or not (–) expressed in normal skin (( figure 5A, B and C) ).

TLR2, TLR4 and TLR9 expression in inflammatory diseases ((figures 1-3)

The expression of these three TLRs was negative (–) or weak (+) in atopic dermatitis or psoriasis. TLR2 and TLR9 were weakly (+) expressed in 3 of 5 biopsies and in the two others the expression was negative (–). The expression of TLR4 was weak (+) in 2 out of 5 biopsies of atopic dermatitis and in the three others the expression was negative (–). In psoriasis skin, TLR2 and TLR4 were negative (–) in 4 of 5 biopsies and in the last the expression was weak (+). TLR9 expression was negative (–) in the five biopsies.

Parapsoriasis and CTCL

TLR2, TLR4 and TLR9 expression in parapsoriasis skin (figures 1-3)

TLR2 and TLR4 expression was negative (–) in 5 out of 6 biopsies and in the last the expression was weak (+). TLR9 expression was negative (–) in 4 out of 6 biopsies and in the two other the expression was weak (+).

TLR2, TLR4 and TLR9 expression in epidermotropic CTCL

Patterns of expression on cutaneous epidermis of MF for TLR2, TLR4 and TLR9 were similar (( figure 4 )A, B and C). These TLRs were weakly (+) expressed in two third of MF biopsies studied (TLR2: 67%, TLR4: 72% and TLR9: 65%). TLR2, TLR4 and TLR9 were moderately (++) or strongly (+++) expressed in the last third of MF epidermis studied. Remarkably, TLR2, TLR4 and TLR9 expression was found in the totality of MF epidermis in our study (( figure 5 )D, E and F). The staining of these TLRs was localized on intermediate and upper layers.

By double immunostaining on frozen sections of MF skin, we observed that T-cell CD3+ ( (figure 6B) ) or CD4+ ( (figure 6A) ) and Langerhans cells CD1a+ ( (figure 6C) ) in red infiltrated the epidermis. TLR2 (in green) was not expressed by the T cell infiltrate (CD3+ or CD4+) but was expressed by keratinocytes. Furthermore, a majority of skin LCs (CD1a+) seem to express TLR2.

Patterns of expression of TLR2, TLR4 and TLR9 on cutaneous SS sections were comparable ( (figure 4D, E and F) ). TLR2, TLR4 and TLR9 were not (–) expressed in one third of SS skin. TLR2, TLR4 and TLR9 were weakly (+) expressed in a second third (37%, 42% and 47%) of SS skin. TLR2, TLR4 and TLR9 were moderately (++) expressed in the last third (32%, 37% and 26%) of SS skin.

Remarkably, only TLR2 was strongly expressed in 5% of SS epidermis (( figure 5 ) G, H and I). The staining of these TLRs was localized on intermediate and upper layers.Thus, MF and SS epidermis displayed very different patterns for TLR2, TLR4 and TLR9 expression: these TLRs were expressed in every MF epidermis while a third of SS epidermis did not express TLR2, TLR4 and TLR9.

Discussion

By immunohistochemistry, we observed that in keratinocytes of inflammatory skin diseases (atopic dermatitis, psoriasis), the expression of TLR2, TLR4 and TLR9 was not increased compared to normal skin. Our results are similar to those obtained by Curry et al. [18] on normal skin, the authors found that TLR2 was weakly expressed by basal layer keratinocytes, that TLR4 was focally expressed by mid epidermal keratinocytes with no expression by basal layer keratinocytes and that TLR9 was weakly or not expressed by epidermis and dermis. Concerning atopic dermatitis, it has been shown that the innate immune recognition of Staphylococcus aureus is defective in atopic dermatitis [19]. The authors hypothesize that the weak human β-defensin-2 (hBD-2) expression in atopic dermatitis may be due to a defect in pattern recognition receptor PRR(s) for S. aureus and they suggest TLR2 as a potential candidate. On psoriatic skin, the basal layers keratinocytes strongly expressed TLR1 but not TLR2, TLR4 and TLR9. However another group [20], has shown that epidermal keratinocytes in normal skin constitutively expressed TLR2 while TLR4 was, in most cases, barely detectable. In contrast, in lesional epidermis from patients with psoriasis, TLR2 was more highly expressed on the keratinocytes of the upper epidermis than on the basal layer and TLR4 expression was similar to that observed in normal skin. In parapsoriasis the expression of the three TLRs was also weak and not significantly different from normal skin. By contrast, in MF skin, we observed an increase in the expression of the three TLRs by keratinocytes. Thus, TLR2 and TLR4 (but not TLR9) are increased according with MF stage (data not shown). Concerning SS, the expression of the three TLRs by keratinocytes was intermediate between inflammatory lesions and MF. A possible correlation could be made with a CTCL Th profile. Indeed Saed G et al. [21], demonstrated that the cutaneous lesions of MF are characterized by an epidermal Th1-type cytokine profile, whereas both blood and skin of patients with SS are characterized by a Th2 profile. Schnare M et al. [22], showed that activation of the adaptive immune response and induction of Th1 effectors requires TLR mediated recognition and signaling whereas Th2 effector responses appear to be independent of TLR function.

Activation of TLRs and thereafter NF-κB and other downstream effectors leads to the production of proinflammatory cytokines and chemokines [23]. TLR2, TLR4 and TLR9 trigger the MyD88 dependent pathway and involve the early phase of NF-κB activation, which leads to the production of inflammatory cytokines. But TLR4 may also trigger the MyD 88 independent pathway and activate interferon (INF) – regulator factor (IRF-3) and thus involve the late phase of NF-κB activation, both of them leading to the production of INFβ and the expression of INF- inducible genes.

By immunohistochemical staining on paraffin-embedded MF specimens, Izban et al. [24] showed that a constitutive activation of NF-κB is not only a feature of CTCL cell- lines, but also of neoplastic T lymphocytes of MF skin. The study was performed on specimens without previous therapy. Interestingly, in most of these cases, keratinocytes also showed an activation of NF-κB p65 (RelA). The authors thus concluded that, this finding may be related to the stimulation of NF-κB activation in keratinocytes in response to various stimulatory cytokines produced by neoplastic MF cells.

Our results supply complementary information: the stimulation of NF-κB activation observed in keratinocytes could be triggered by TLR2, TLR4 and TLR9 activation. Indeed we show that the expression of the three TLRs by the keratinocytes is increased in MF. Thus, the development of the skin lesions in MF appears associated to an increase of TLR2, TLR4 and TLR9 expression by keratinocytes in cutaneous lesions. Furthermore, by double labelling, we checked that T-cells (CD3+ or CD4+) were not responsible for the increase TLR2 in expression ( (figure 6A and B) ). As a majority of skin LCs seems to express TLR2 ( (figure 6C) ) we do not exclude that LCs could partially participate at the increase of TLR2 expression in MF epidermis.

Moreover, TLR2, TLR4, and TLR9 can be activated by viral antigens [16]. Thus, TLR4 is activated by respiratory syncytial viruses (RSV) and retrovirus infection [25]. TLR2 and TLR9 have been described as activated by viruses of the Herpesviridae family [26, 27]. Herpes simplex virus (HSV-1 and HSV-2) are common human pathogens. Kurt-Jones et al. demonstrated that TLR2 mediates the induction of inflammatory cytokines in response to HSV-1, and surprisingly, its role in alerting the innate response has pathological rather than protective consequences [26].Herpes is a family of viruses which can cause chronic inflammation disease that may be related to TLR2, TLR4 and TLR9 activation by molecular pattern on Herpes virus [25-28]. It has been shown in our laboratory that EBV proteins were expressed in keratinocytes in the epidermis of CTCL patients [29] and recently, the presence of EBV in Langerhans cells of CTCL lesions has also been demonstrated [30]. In addition, it is well known that patients with epidermotropic CTCL often develop more severe and frequent cutaneous herpes infection. Eliopoulos et al. [31] showed that stable or transient LMP1 (latent membrane protein 1) expression in SV 40 transformed keratinocytes induced significant levels of IL6 expression and secretion via the NF-κB mechanism.

According to the results of our study, the following hypothesis about the inflammatory reaction observed in the skin of MF patients may be raised. Herpes viruses (HSV, EBV) could trigger TLR2, TLR4 and TLR9, the expression of which is increased in keratinocytes of epidermotropic CTCL leading to NF-κB activation. The activation of TLR pathways leads to pro-inflammatory cytokine secretions that could contribute to maintain the chronic activation of the CD4+ T-cell infiltrates in CTCL skin lesions.

In conclusion, we demonstrate that the expression of TLRs 2, 4, 9 is increased in epidermal lesion of MF. Their activation could play a role in the induction and maintenance of the T-lymphocytes in infiltrate cutaneous lesions of MF and SS.

References

1 Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000; 406: 782-7.

2 Zhang D, Zhang G, Hayden MS, Greenblatt MB, Bussey C, Flavell RA, Ghosh S. A toll-like receptor that prevents infection by uropathogenic bacteria. Science 2004; 303: 1522-6.

3 Kim J. Review of the innate immune response in acne vulgaris: activation of Toll-like receptor 2 in acne triggers inflammatory cytokine responses. Dermatology 2005; 211: 193-8.

4 Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004; 4: 499-511.

5 Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004; 16: 3-9.

6 Song PI, Abraham TA, Park Y, Zivony AS, Harten B, Edelhauser HF, Ward SL, Armstrong CA, Ansel JC. The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells. Invest Ophthalmol Vis Sci 2001; 42: 2867-77.

7 Pivarcsi A, Bodai L, Rethi B, Kenderessy-Szabo A, Koreck A, Szell M, Beer Z, Bata-Csorgoo Z, Magocsi M, Rajnavolgyi E, Dobozy A, Kemeny L. Expression and function of Toll-like receptors 2 and 4 in human keratinocytes. Int Immunol 2003; 15: 721-30.

8 Kawai K, Shimura H, Minagawa M, Ito A, Tomiyama K, Ito M. Expression of functional Toll-like receptor 2 on human epidermal keratinocytes. J Dermatol Sci 2002; 30: 185-94.

9 Kikuchi A, Naka W, Harada T, Sakuraoka K, Harada R, Nishikawa T. Parapsoriasis en plaques: its potential for progression to malignant lymphoma. J Am Acad Dermatol 1993; 29: 419-22.

10 Simon M, Flaig MJ, Kind P, Sander CA, Kaudewitz P. Large plaque parapsoriasis: clinical and genotypic correlations. J Cutan Pathol 2000; 27: 57-60.

11 Vakeva L, Sarna S, Vaalasti A, Pukkala E, Kariniemi AL, Ranki A. A retrospective study of the probability of the evolution of parapsoriasis en plaques into mycosis fungoides. Acta Derm Venereol 2005: 318-23.

12 Pimpinelli N, Olsen EA, Santucci M, Vonderheid E, Haeffner AC, Stevens S, Burg G, Cerroni L, Dreno B, Glusac E, Guitart J, Heald PW, Kempf W, Knobler R, Lessin S, Sander C, Smoller BS, Telang G, Whittaker S, Iwatsuki K, Obitz E, Takigawa M, Turner ML, Wood GS. Defining early mycosis fungoides. J Am Acad Dermatol 2005: 1053-63.

13 Pancake BA, Zucker-Franklin D, Coutavas EE. The cutaneous T cell lymphoma, mycosis fungoides, is a human T cell lymphotropic virus-associated disease. A study of 50 patients. J Clin Invest 1995; 95: 547-54.

14 Papadavid E, Economidou J, Psarra A, Kapsimali V, Mantzana V, Antoniou C, Limas K, Stratigos A, Stavrianeas N, Avgerinou G, Katsambas A. The relevance of peripheral blood T-helper 1 and 2 cytokine pattern in the evaluation of patients with mycosis fungoides and Sezary syndrome. Br J Dermatol 2003; 148: 709-18.

15 Jumbou O, Mollat C. N’Guyen JM, Billaudel S, Litoux P, Dreno B. Increased anti-Epstein-Barr virus antibodies in epidermotropic cutaneous T-cell lymphoma: a study of 64 patients. Br J Dermatol 1997; 136: 212-6.

16 Morrison LA. The Toll of herpes simplex virus infection. Trends Microbiol 2004; 12: 353-6.

17 Pimpinelli N, Olsen EA, Santucci M, Vonderheid E, Haeffner AC, Stevens S, Burg G, Cerroni L, Dreno B, Glusac E, Guitart J, Heald PW, Kempf W, Knobler R, Lessin S, Sander C, Smoller BS, Telang G, Whittaker S, Iwatsuki K, Obitz E, Takigawa M, Turner ML, Wood GS. Defining early mycosis fungoides. J Am Acad Dermatol 2005; 53: 1053-63.

18 Curry JL, Qin JZ, Bonish B, Carrick R, Bacon P, Panella J, Robinson J, Nickoloff BJ. Innate immune-related receptors in normal and psoriatic skin. Arch Pathol Lab Med 2003; 127: 178-86.

19 Ong PY. Is/are pattern recognition receptor(s) for Staphylococcus aureus defective in atopic dermatitis? Dermatology 2006; 212: 19-22.

20 Baker BS, Ovigne JM, Powles AV, Corcoran S, Fry L. Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis. Br J Dermatol 2003; 148: 670-9.

21 Saed G, Fivenson DP, Naidu Y, Nickoloff BJ. Mycosis fungoides exhibits a Th1-type cell-mediated cytokine profile whereas Sezary syndrome expresses a Th2-type profile. J Invest Dermatol 1994; 103: 29-33.

22 Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2001; 2: 947-50.

23 Li ZW, Rickert RC, Karin M. Genetic dissection of antigen receptor induced-NF-kappaB activation. Mol Immunol 2004; 41(6-7): 701-14.

24 Izban KF, Ergin M, Qin JZ, Martinez RL, Pooley RJ, Saeed S, Alkan S. Constitutive expression of NF-kappa B is a characteristic feature of mycosis fungoides: implications for apoptosis resistance and pathogenesis. Hum Pathol 2000; 31: 1482-90.

25 Haynes LM, Moore DD, Kurt-Jones EA, Finberg RW, Anderson LJ, Tripp RA. Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus. J Virol 2001; 75: 10730-7.

26 Kurt-Jones EA, Chan M, Zhou S, Wang J, Reed G, Bronson R, Arnold MM, Knipe DM, Finberg RW. Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis. Proc Natl Acad Sci USA 2004; 101: 1315-20.

27 Lund J, Sato A, Akira S, Medzhitov R, Iwasaki A. Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J Exp Med 2003; 198: 513-20.

28 Krug A, Luker GD, Barchet W, Leib DA, Akira S, Colonna M. Herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9. Blood 2004; 103: 1433-7.

29 Dreno B, Celerier P, Fleischmann M, Bureau B, Litoux P. Presence of Epstein-Barr virus in cutaneous lesions of mycosis fungoides and Sezary syndrome. Acta Derm Venereol 1994; 74: 355-7.

30 Knol AC, Quereux G, Pandolfino MC, Khammari A, Dreno B. Presence of Epstein-Barr Virus in Langerhans Cells of CTCL Lesions. J Invest Dermatol 2005; 124: 280-2.

31 Eliopoulos AG, Stack M, Dawson CW, Kaye KM, Hodgkin L, Sihota S, Rowe M, Young LS. Epstein-Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-kappaB pathway involving TNF receptor-associated factors. Oncogene 1997; 14: 2899-916.