Certain herpesviruses and poxviruses are known to encode chemokine homologues, and 13 viral chemokine homologues have been identified (1
). Most viral chemokines are broad-spectrum chemokine receptor antagonists which are likely to inhibit cellular infiltrates in virus-infected tissues. Notably, however, vMIP-I, vMIP-II, and vMIP-III, encoded by Kaposi's sarcoma-related herpesvirus (KSHV)-human herpesvirus 8, are chemokine receptor agonists; vMIP-I and vMIP-II are the agonists for CCR8, while vMIP-III is the agonist for CCR4 (1
). Since CCR4 and CCR8 are known to be selectively expressed by Th2 cells and immunosuppressive regulatory T cells (18
), tissues infected with KSHV may preferentially attract these types of T-cell subsets via vMIP-I, vMIP-II, and vMIP-III. Consistently, Sozzani et al. demonstrated that T cells infiltrating KSHV-infected tissues predominantly secreted Th2-type cytokines (37
). Since Th1 cells play the major roles in host immune responses against virus-infected cells, selective accumulation of Th2 cells and local production of Th2-type cytokines in virus-infected tissues may be advantageous for the viruses by downregulating host antiviral Th1 responses. In keeping with this notion, several viruses also encode Th2-cytokine homologues and Th1-cytokine inhibitors; EBV encodes an interleukin-10 (IL-10) homologue, KSHV encodes vIL-6 and the gamma interferon inhibitor vIRF-1, and myxoma virus encodes the gamma interferon inhibitor M-T7 (1
TARC/CCL17 and MDC/CCL22 are the natural ligands of CCR4, while I-309/CCL1 is the ligand of CCR8 (45
). Here, we have demonstrated that EBV-immortalized B cells selectively express the genes for TARC/CCL17, MDC/CCL22, and I-309/CCL1 (Fig. ) and secrete these chemokines, especially MDC/CCL22, in large quantities (Fig. ). Furthermore, EBV infection induced peripheral-blood B cells to express TARC/CCL17 and MDC/CCL22 in parallel with the expression of LMP1 (Fig. ). We further showed that (i) LMP1 induces the expression of TARC/CCL17 and MDC/CCL22 in BJAB cells (Fig. ), (ii) the inhibitors of the TRAF/NF-κB pathway and the p38/ATF2 pathway effectively suppress the expression of TARC/CCL17 and MDC/CCL22 in EBV-immortalized B cells and LMP1-transfected BJAB cells (Fig. ), and (iii) two proximal NF-κB sites and a single AP-1 site in the promoter region of MDC/CCL22 are critically involved in the activation of the MDC/CCL22 promoter by LMP1 (Fig. ). Collectively, LMP1, which is known to induce its target genes via signaling pathways involving TRAF/NF-κB, JNK/AP-1, p38/ATF2, and JAK3/STAT (10
), is likely to induce the gene expression of TARC/CCL17 and MDC/CCL22 in EBV-infected B cells via activation of NF-κB and most probably ATF2. Currently, LMP1 is known to induce the B-cell activation markers CD23 and CD40, adhesion molecules such as LFA-1 and ICAM-1, antiapoptotic molecules such as bcl-2 and A20, matrix metalloproteinase 9, and CD83 (9
). Thus, the list of cellular genes induced by LMP1 now includes the chemokines TARC/CCL17 and MDC/CCL22.
Even though the inhibitor of the p38/ATF2 pathway efficiently suppressed the expression of TARC/CCL17 and MDC/CCL22 in EBV-immortalized B cells and LMP1-transfected BJAB cells (Fig. ), we were unable to find any potential ATF2 site in the TARC/CCL17 and MDC/CCL22 promoter regions (−819 to −22 and −722 to −11, respectively) (Fig. ). In addition, a single AP-1 site in the proximal promoter region of MDC/CCL22 was necessary for the full activation of the MDC/CCL22 promoter (Fig. ), even though the inhibitor of the JNK/AP-1 pathway did not suppress the expression of TARC/CCL17 and MDC/CCL22 in EBV-immortalized B cells and LMP1-transfected BJAB cells (Fig. ). In this context, ATF2 was shown to activate the promoters of IL-8/CXCL8 and c-Jun via the AP-1 site (10
). Thus, ATF2 is likely to be involved in the promoter activation of TARC/CCL17 and MDC/CCL22 by targeting the AP-1 site.
Even though we were unable to directly demonstrate the activation of the TARC/CCL17 promoter (−819 to −22) by LMP1 under the present experimental conditions, we have also found potential NF-κB and AP-1 sites in the TARC/CCL17 promoter region (−819 to −22) in an arrangement similar to that of MDC/CCL22 (Fig. ). Thus, these NF-κB and AP-1 sites may also be responsible for the induction of TARC/CCL17 by LMP1. On the other hand, I-309/CCL1 and MEC/CCL28 were not directly induced by EBV (Fig. and ), and their expression in EBV-immortalized B cells was not affected by various signaling inhibitors (Fig. ). Thus, their expression may be due to the plasmablast-like stage of differentiation of EBV-immortalized B cells. Indeed, we have consistently observed the expression of I-309/CCL1 and MEC/CCL28 in human myeloma cell lines (data not shown). Since TARC/CCL17 and MDC/CCL22 are known to selectively attract Th2 cells and immunosuppressive regulatory T cells via CCR4 (18
), EBV may induce host B cells to produce these chemokines to preferentially attract Th2 cells and immunosuppressive regulatory T cells. This may be advantageous for the survival of EBV-infected B cells by shifting local immune responses to Th2. In addition, Th2-type cytokines, such as IL-4, IL-5, IL-6, and IL-10, are also known to promote the growth of EBV-immortalized B cells (23
The repertoire of chemokines produced by EBV-immortalized B cells is not, however, so straightforward. While resting peripheral-blood B cells constitutively express MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5 (all CCR5 ligands), EBV-immortalized B cells further upregulate their expression (Fig. ) and secrete these chemokines, especially MIP-1α/CCL3 and MIP-1β/CCL4, in large quantities (Fig. ). EBV infection of purified B cells also rapidly upregulates the expression of these chemokines (Fig. ). However, it appears that EBV is not directly responsible for the upregulation of these chemokines (Fig. ). Furthermore, in contrast to TARC/CCL17 and MDC/CCL22, their expression is mainly dependent on signaling pathways, such as the MEK/ERK and JAK3/STAT pathways (Fig. ). Since LMP1 and LMP2A provide a surrogate T-cell help and a surrogate antigen receptor signal, respectively (5
), EBV-immortalized B cells are phenotypically activated B cells. Thus, the upregulation of MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5 in EBV-immortalized B cells may be related to the activated phenotype of B cells imposed by EBV infection. In agreement with this idea, previous studies have shown that the MEK/ERK and JAK3/STAT pathways are involved in signals for B-cell activation (8
), and activated B cells selectively produce MIP-1α/CCL3 and MIP-1β/CCL4 (4
MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5 produced by EBV-infected B cells are likely to attract Th1 cells and activated cytotoxic T cells via CCR5 (19
). Thus, EBV-infected B cells may paradoxically promote their own elimination by the host immune system. However, this may be beneficial for the eventual survival of EBV by self-limiting its highly tumorigenic latency III infection and establishing persistent infection in a pool of memory B cells with a more restricted pattern of expression of latent proteins (41
). Interestingly, however, Bystry et al. have recently demonstrated that activated B cells produce MIP-1α/CCL3 and MIP-1β/CCL4 and attract regulatory T cells in naive mice (4
). Similarly, therefore, the production of MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5 by EBV-infected B cells may initially attract regulatory T cells instead of Th1 cells and cytotoxic T cells in naive human hosts.
In conclusion, we have elucidated the expression profile of chemokines in EBV-immortalized B cells. In particular, EBV-immortalized B cells produce TARC/CCL17, MDC/CCL22, MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5 in substantial-to-large quantities. The upregulation of TARC/CCL17 and MDC/CCL22 is mainly induced by LMP1, while that of MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5 is likely to be due to the activated-B-cell phenotype of EBV-infected B cells. Consistently, the serum MDC/CCL22 levels were significantly elevated in IM patients (Fig. ). The production of these chemokines by EBV-infected B cells may variably affect the virus-host relationship and may account for some features of IM and other EBV-associated diseases.