The Viral Chemokine Macrophage Inflammatory Protein-II Is a Selective Th2 Chemoattractant

HUMAN HERPES VIRUS 8 (HHV8), also known as Kaposi’s sarcoma virus, is associated with Kaposi’s sarcoma (KS), body cavity–based lymphoma, and Castelman’s disease.1-4The HHV8 genome includes three open reading frames coding for proteins with considerable (∼40%) identity to human CC chemokines and one coding for a chemokine receptor, ORF74.5 KS is an opportunistic tumor characterized by prominent angiogenesis and leukocyte infiltration,6 7 including T cells and monocytes.

The current concept of the multistep process of leukocyte recruitment into tissues envisions chemotactic agonists as one of the key effector molecules.8-10 Chemokines are a superfamily of chemotactic proteins that can be divided in four groups on the basis of a cysteine structural motif. Most of the chemokines fall in two subfamilies: the α (or CXC) chemokines, mainly active on neutrophils and lymphocytes and the β (or CC) chemokines active on multiple subsets of mononuclear cells. Lymphotactin (γ or C chemokines) and fractalkine (δ or CX3C chemokines) may define two additional groups of this superfamily.10,11 Recent results indicate that polarized T helper 1 (Th1) and Th2 populations differentially express chemokine receptors and respond to chemotactic agonists.12 13 

Here we show that CD4 and CD8 cells infiltrating KS have predominantly a type II cytokine profile and that the KS chemokine viral macrophage inflammatory protein-II (vMIP-II) is a selective attractant for type II T cells and interacts as an agonist with a receptor (CCR8) selectively expressed on this polarized subset.

Human monocytes and neutrophils were obtained from buffy coats of healthy blood donors by density gradients on Ficoll (Biochrom) Percoll (Pharmacia, Uppsala, Sweden), as previously described.14Monocyte-derived dendritic cells (mono-DC) and CD34-derived dendritic cells (CD34-DC) were obtained as previously described.15Stable transfectants of CCR8 (TER1) were prepared by electroporation of Jurkat cells with pcDNA3-HA/TER1 and subsequent selection in G418.16 Th1 and Th2 cultures were obtained as previously described.13 

Cell migration was evaluated using a chemotaxis microchamber technique as previously described.14 Monocytes, neutrophils, DCs, and Jurkat cells were tested using a 5-μm pore-size polycarbonate filter. For Jurkat cells filters were previously coated with murine collagen type IV. At the end of the incubation, filters were removed, stained with Diff-Quik (Baxter s.p.a., Rome, Italy), and five high-power oil-immersion fields were counted. T-cell cultures were tested with the leading front methods using nitrocellulose filters, and migration was evaluated as distance (μm) migrated by the two fastest cells.14 Human recombinant MCP-3 (MCP-3) was a kind gift of Dr A. Minty (Sanofi Elf Bio Recherches, Labège, France). vMIP-II was chemically synthesized as previously described17 and was a kind gift of Dr Ian Clark-Lewis (University of British Columbia, Vancouver, Canada).

Generation of T helper cell lines was performed by stimulating cord blood mononuclear cells with 2 μg/mL PHA (Wellcome, Beckenham, UK) in polarizing conditions as described.18 Differentiation of Th1 cells was obtained by addition of 2 ng/mL of IL-12 and 1,000 U/mL of interferon-α (IFN-γ; Hoffmann-La Roche Inc, Nutley, NJ) together with 200 ng/mL of neutralizing anti–interleukin-4 (IL-4) antibodies (Pharmingen, San Diego, CA), whereas differentiation of Th2 cells was obtained by addition of 200 U/mL of IL-4 (Pharmingen) together with 2 μg/mL of neutralizing anti–IL-12 antibodies 17F7 and 20C2 (a gift of M. Gately, Hoffmann-La Roche Inc). Cells were evaluated for their cytokine production profiles by intracellular staining as previously described.13 Polarized CD4⁺ Th1 and Th2 cells or CD8⁺ Tc1 and Tc2 cells were separated by immunomagnetic negative selection by incubating the cells with anti-CD4 or anti-CD8 monoclonal antibodies (Pharmingen).

Skin biopsy specimens were incubated in IL-2 (20 U/mL)-containing medium for 7 to 10 days to expand in vivo–activated IL-2 receptor–expressing T cells as previously described.19T-cell suspensions obtained from each skin specimen were then cloned under limiting dilution conditions (0.5 cell/well) in the presence of phytohemagglutinin (PHA; 1% vol/vol), IL-2 (20 U/mL), and irradiated peripheral blood mononuclear cells (PBMC) as feeder cells. The cell surface phenotype of clonal T-cell blasts was assessed with flow cytometry by using fluorescein isothiocyanate (FITC)-conjugated anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies (MoAbs). T blasts (1 × 10⁶/mL) from each clone were then stimulated with PMA plus ionomycin for 24 hours, and the production of IL-4 and IFN-γ was measured in cell-free culture supernatants by using appropriate enzyme-linked immunosorbent assay (ELISA) assays.

T-cell clones were generated under the same experimental conditions from skin biopsy specimens of one normal volunteer, two patients with alopecia areata, three patients with atopic dermatitis, and three patients with KS. Higher proportions of CD8⁺ than CD4⁺ T-cell clones were generated from the skin of KS patients (369 and 53 clones, respectively), whereas CD4⁺T-cell clones were prevalent in controls (53 and 14 clones for CD4⁺ and CD8⁺, respectively). As reported in Fig 1, the majority of CD4⁺T-cell clones generated from healthy skin or the skin of alopecia areata patients showed a Th1-skewed profile, whereas those generated from the skin of atopic dermatitis patients showed a more heterogeneous cytokine profile. However, virtually no CD8⁺ T-cell clones with Tc2 profile, except in one patient with atopic dermatitis, were found. By contrast, high proportions of both CD4⁺ and CD8⁺ T-cell clones generated from the skin of KS patients showed a Th2-skewed phenotype (Fig 1).

The capacity of vMIP-II to elicit directional migration of various leukocyte populations was then studied (Fig2). vMIP-II induced migration of human monocytes with an ED50 of 32 ± 4 ng/mL (∼3 nmol/L) and maximal activity at 100 ng/mL. Under the same conditions MCP-3, used as reference chemoattractant, had an ED50 of 15 ± 3, and the maximal response was 1.9 ± 0.3-fold (n = 3) higher than that of vMIP-II. Checkerboard analysis showed that vMIP-II elicited actual chemotactic migration in monocytes. This contrasts with previous data17 in which vMIP-II was a weak monocyte attractant. This difference in vMIP-II efficacy is presumably due to the cell preparation and assay used. Other leukocyte populations, neutrophils, monocyte-derived or CD34-derived dendritic cells (Fig 2), NK cells, unseparated lymphocytes, and naive cord blood T cells (data not shown) showed no substantial response to vMIP-II.

vMIP-II showed weak chemotactic activity for IL-2–activated, but not resting, T cells (data not shown). These indications prompted a more careful analysis of the capacity of vMIP-II to attract T-lymphocyte subpopulations. As shown in Fig 2A, vMIP-II showed preferential attraction of Th2 versus Th1 cells, both in bulk cultures and in clonal populations. It has recently been shown that chemokine receptors are differentially expressed in Th1 versus Th2 cells.12,13,16,20-22 CCR4 and CCR3 are expressed predominantly in Th2 cells, whereas Th1 cells express CCR5 and CXCR3.13 A similar differential expression of chemokine receptors has been observed recently in CD8⁺ T cells with different cytokine profiles23 (and unpublished results).

vMIP-II has been shown to interact with multiple chemokine receptors as an antagonist or as an agonist.17,24 It binds CCR3,24 a receptor expressed preferentially on polarized Th2 cells,12,13,21,22 and shows activity on eosinophils.24 As shown in Fig 2, vMIP-II interacts also with CCR8, the I309 receptor,16,25 26 and elicits migration of CCR8-transfected cells (J/CCR8; Fig 2A). Chemotactic index of J/CCR8 for vMIP-II was 5.3 ± 1.5 (n = 5) compared with 2.8 (n = 2) for I-309 (at the peak concentrations of 100 ng/mL and 30 ng/mL, respectively). Chemotactic index to SDF-1 (1 μg/mL), used as reference chemoattractant, through its endogenous receptor (CXCR4), was 4.1 ± 0.3 (n = 4). As shown in Fig 2C, CCR8 was predominantly expressed in CD4⁺ Th2 cells and in CD8 T cells with a Th2 phenotype (Tc2). Hence, the ability of vMIP-II to act on chemokine receptors, such as CCR8 that is expressed on Th2 cells, underlies the ability of vMIP-II to selectively attract these cells.

Both CD4⁺ and CD8⁺ T cells comprise of different subsets based on the cytokines they produce.27,28Th1 cells predominantly mediate phagocyte-dependent protective immunity as well as inflammatory autoimmune disorders, whereas Th2 cells are responsible for phagocyte-independent protection and are prominent in the pathogenesis of allergic diseases.28 The results presented here show that KS lesions are infiltrated by CD8⁺and CD4⁺ T cells with a predominant type II cytokine profile. The viral chemokine encoded by KS-associated HHV8 was a selective attractant for type II T cells and showed agonist activity for CCR8, a receptor selectively expressed on polarized type II T cells. There is evidence that activation of a Th2 response can lead to delayed virus clearance.29 Hence, the capacity of the HHV8-encoded chemokine vMIP-II to selectively attract monocytes and Th2 cells is likely a component of the virus strategy to subvert immunity.