Eta-1 (Osteopontin): An Early Component of Type-1 (Cell-Mediated) Immunity Samy Ashkar, 1 * Georg F. Weber, 2,3 * Vassiliki Panoutsakopoulou, 2,4 Marie E. Sanchirico, 2,4 Marianne Jansson, 2,4 Samer Zawaideh, 1 Susan R. Rittling, 5 David T. Denhardt, 5 Melvin J. Glimcher, 1 Harvey Cantor 2,4 ‡ Cell-mediated (type-1) immunity is necessary for immune protection against most intracellular pathogens and, when excessive, can mediate organ-specific autoimmune destruction. Mice deficient in Eta-1 (also called osteopontin) gene expression have severely impaired type-1 immunity to viral infection [herpes simplex virus–type 1 (KOS strain)] and bacterial infection (Listeria monocyto- genes) and do not develop sarcoid-type granulomas. Interleukin-12 (IL-12) and interferon-g production is diminished, and IL-10 production is increased. A phosphorylation-dependent interaction between the amino-terminal portion of Eta-1 and its integrin receptor stimulated IL-12 expression, whereas a phos- phorylation-independent interaction with CD44 inhibited IL-10 expression. These findings identify Eta-1 as a key cytokine that sets the stage for efficient type-1 immune responses through differential regulation of macrophage IL-12 and IL-10 cytokine expression. The development of cell-mediated (type-1) im- mune responses is necessary for protection against the growth of many infectious pathogens and, when excessive, can mediate autoimmune host tissue destruction. Although macrophage activation by microbial pathogens (1, 2) and foreign body reactions (3) are associated with type-1 immunity, the cellular and molecular events that imprint this response are not fully understood. An essential early step in this pro- cess is macrophage production of IL-12 at sites of infection, whereas early IL-10 production inhibits this response (4 ). Although IL-12 re- sponses can be triggered by an interaction be- tween the CD40 ligand on activated T cells and CD40 on macrophages (4 ), this interaction also induces the inhibitory IL-10 cytokine (5, 6 ), and its transient nature may not suffice for sustained IL-12 induction in vitro (7 ) or in vivo (8). A gene product that may play an impor- tant role in the development of type-1 immu- nity is the T cell cytokine Eta-1 (for early T lymphocyte activation–1), also known as os- teopontin (Opn) (9). The Eta-1 gene is ex- pressed in T cells early in the course of bacterial infections (within 48 hours), and interaction of its protein product with macro- phages can induce inflammatory responses (10). Genetic resistance to infection by cer- tain strains of Rickettsia may depend on Eta- 1– dependent attraction of monocytes into in- fectious sites and acquisition of bacteriocidal activity (11); the granulomatous responses characteristic of sarcoidosis and tuberculosis are associated with high levels of Eta-1 ex- pression (12). Granuloma formation in these human dis- eases is a cellular consequence of type-1 immu- nity (12), and sarcoid-type granulomas can be induced in mice after injection of polyvinyl pyrrolidone (PVP) (13). Because certain murine models of parasite-induced granulomas may reflect a mixture of type-2 and type-1 immunity (6 ), we first established the importance of IL- 12– dependent type-1 immunity in this murine model of granuloma formation. An intense granulomatous response was provoked shortly after (subcutaneous) injection of PVP into C57BL/6 (1/1) but not C57BL/6 nu/nu strains of mice. This response was diminished by 70 to 80% in C57BL/6 IL-12 2/2 mice and was en- hanced two- to threefold in C57BL/6 IL-10 2/2 mice (Fig. 1, A and B). Because C57BL/6 nu/nu mice coinjected with PVP and puri- fied Eta-1 displayed a granulomatous reac- tion, this gene product can partially substi- tute for activated T lymphocytes in this setting (Fig. 1, A and B). We then asked whether mice deficient in Eta-1 secondary to targeted gene mutation (14 ) formed granulomas after PVP injection. Eta- 1 2/2 mice did not develop a detectable granu- lomatous response after challenge with PVP; the response was partially restored by coinjection of purified Eta-1 with PVP (Fig. 1, A and B). Histologic analysis of granulomas formed in Eta-1 1/1 mice and in Eta-1 2/2 mice reconsti- tuted with purified Eta-1 revealed a similar mac- rophage-dominant cellular infiltrate: About 85% of granulomatous cells in both cases were Mac- 1 1 , whereas 5 to 10% were CD3 1 T cells or B220 1 B cells. BP-55 1 neutrophils, which were only a minor component (1 to 2%) of granulo- mas in these mice, increased 5- to 10-fold in the granulomas of IL-10 2/2 mice (Fig. 1C). Eta- 1 2/2 mice also displayed defective granuloma- tous responses to injection of collagen and latex, consistent with reports that human T cells resi- dent in sterile granulomas have high expression of Eta-1 (12). Restimulation of lymph nodes draining subcutaneous sites of PVP injection in Eta-1 2/2 mice and control mice with PVP re- vealed impaired IL-12 and interferon-g (IFN-g) responses: The IL-12 response was reduced by ;95%, and the IFN-g response of Eta-1 2/2 mice was reduced by 90% in comparison to Eta-1 1/1 controls (Fig. 1D). We next defined the role of Eta-1 in the immune response to herpes simplex virus–type 1 (HSV-1) (KOS strain) infection. Eta-1 2/2 mice infected by HSV-1 [4 3 10 6 plaque- forming units (PFU) via the cornea] did not develop a significant tuberculin-type delayed- type hypersensitivity (DTH) response after footpad challenge with HSV-1 (10 5 PFU), in contrast to the strong DTH response of Eta- 1 1/1 controls (Fig. 2A, left). Although the numbers of T cells and proportions of T cell subsets in the thymus and peripheral lymphoid tissues of Eta-1 2/2 mice were similar to Eta- 1 1/1 littermates (15), defective antiviral DTH responses might reflect a subtle alteration in lymphocyte or macrophage development. We therefore tested the effects of acute in vivo depletion of Eta-1 with a neutralizing antibody. Administration of antibody to Eta-1 (LF-123) (16 ) immediately before and repeatedly after HSV-1 infection efficiently inhibited the DTH response upon rechallenge (Fig. 2A, right). Corneal HSV-1 infection can also lead to a destructive type-1 autoimmune inflammatory reaction, herpes simplex keratitis (HSK), initi- ated by CD4 cells that recognize a viral peptide mimic (17 ). This inflammatory response de- pends on the production of IL-12 and is inhib- ited by IL-10 (18). Within 10 to 14 days after corneal HSV-1 infection, ;65% of control Eta- 1 1/1 mice developed HSK, whereas HSV-1– infected Eta-1 2/2 mice did not readily develop this disease (Fig. 2B). Analysis of cells from the draining lymph nodes of virus-infected Eta- 1 2/2 and Eta-1 1/1 mice indicated that they responded similarly to HSV-1 according to [ 3 H]thymidine incorporation after viral restimu- 1 Laboratory for Skeletal Disorders and Rehabilitation, Department of Orthopedic Surgery, Children’s Hospi- tal, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA. 2 Department of Cancer Im- munology and AIDS, Dana-Farber Cancer Institute; 3 Department of Medicine; 4 Department of Pathology; Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA. 5 Faculty of Arts and Sciences, Division of Life Sciences, Rutgers University, 604 Allison Road, NJ 08854, USA. *These authors contributed equally to this work. Present address: Division of Radiation and Cancer Biology, New England Medical Center, 750 Washing- ton Street, Boston, MA 02111, USA. ‡To whom correspondence should be addressed. E- mail: Harvey_Cantor@DFCI.harvard.edu R EPORTS 4 FEBRUARY 2000 VOL 287 SCIENCE www.sciencemag.org 860