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Homeostatic Regulation of the Immune System by Receptor Tyrosine Kinases of the Tyro 3 Family

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Science  13 Jul 2001:
Vol. 293, Issue 5528, pp. 306-311
DOI: 10.1126/science.1061663

Abstract

Receptor tyrosine kinases and their ligands mediate cell-cell communication and interaction in many organ systems, but have not been known to act in this capacity in the mature immune system. We now provide genetic evidence that three closely related receptor tyrosine kinases, Tyro 3, Axl, and Mer, play an essential immunoregulatory role. Mutant mice that lack these receptors develop a severe lymphoproliferative disorder accompanied by broad-spectrum autoimmunity. These phenotypes are cell nonautonomous with respect to lymphocytes and result from the hyperactivation of antigen-presenting cells in which the three receptors are normally expressed.

The elimination of reactive lymphocytes is a central feature of homeostatic regulation in the immune system. Although clonal expansion of lymphocytes is essential for immune responses, activated T and B cells must be deleted once the antigens that triggered their expansion have been eradicated. Similarly, autoreactive T cell clonotypes pose a severe threat to tissue and organ integrity, and must also be deleted. Deficiencies in the homeostatic regulation of expanded or autoreactive lymphocytes lead to lymphoproliferative disorders, impaired immune function, autoimmunity, and death (1).

In the mature immune system, lymphocyte numbers are under the control of a wide variety of soluble cytokines, as well as cell surface inhibitory and costimulatory molecules. Although many of these regulators bind to receptors that are coupled to cytoplasmic protein-tyrosine kinases (PTKs), none of them is known to signal through the more direct mechanism of binding and activating a receptor with intrinsic PTK activity (2). This notwithstanding, we have found that three structurally related receptor PTKs—Tyro 3 (3, 4), Axl (3, 5), and Mer (3, 6)—play an essential immunoregulatory role. These receptors are, together with their ligands Gas6 and Protein S (7, 8), widely coexpressed in cells of the immune, nervous, vascular, and reproductive systems, but their biological roles in these tissues have only recently begun to be addressed directly (9).

We have analyzed the immune system phenotypes of an allelic series of mice that are singly, doubly, and triply mutant in theTyro 3, Axl, and Mer genes (9). At birth, even triple mutants of this series displayed peripheral lymphoid organs of normal size and weight, and the initial postnatal development of both their lymphoid and myeloid cell lineages was not obviously different from wild type. Beginning at ∼4 weeks, however, the spleens and lymph nodes of these triple mutants grew at elevated rates relative to wild type (Fig. 1, A through C), such that by 1 year of age, their spleen weights were on average 10 times that of wild type (C57Bl/6 × 129sv) (Fig. 1C). Dramatic enlargements of lymph nodes were also evident in all triple mutants, notably in the submaxillary, popliteal, and mesenteric nodal stations (Fig. 1A). These changes were progressively dependent on the inactivation of all three genes in theTyro 3 family (Fig. 1C), a genetic interaction through the allelic series that is consistent with the extensive coexpression of Tyro 3, Axl, and Mer in cells of the immune system and elsewhere (9).

Figure 1

Aberrant lymphocyte proliferation and growth of peripheral lymphoid organs in Tyro 3/Axl/Mer triple mutants. (A) Enlarged submaxillary lymph node (arrow) in a 10-month-old triple mutant female. (B) Relative sizes of the spleen in a triple mutant (TAM) versus wild-type (WT) male at 1 year. (C) Adult spleen weight as a function of genotype. T, A, and M representTyro 3, Axl, and Mer single mutants, respectively, TM represents Tyro 3/Mer double mutants, and so forth. TaM representsTyro3 –/– Axl +/– Mer –/–mice. Average spleen weights (±SEM) are plotted for pooled male and female mice between 5 and 14 months of age; n = 3 (TM), 4 (A, M, TaM), 5 (T), 6 (TA), 7 (AM), 8 (TAM), and 9 mice (WT). (D) FACS scans of expression of the B220 B cell marker versus the TCRαβ T cell marker, in cells from wild-type (WT) and triple mutant (TAM) spleen and lymph node at 2 months of age. In these and all subsequent FACS scan panels, numbers in each quadrant are the percentage of cells in the sort that occupy that quadrant. (E) Expression of the T cell markers CD8 versus CD4 in WT and TAM spleen and lymph node at 2 months of age. (F) Colonies of small, darkly staining lymphocytes (arrows) in the liver, and in (G), the kidney, of a triple mutant. Bar, 0.1 mm.

The aberrant growth of peripheral lymphoid organs was primarily due to the hyperproliferation of B and T cells. Although both classes of lymphocyte were greatly increased in the triple mutants, flow cytometric analyses (10) revealed a modest but selective enrichment of T cells over B cells (Fig. 1, D and E). Within T cell populations, we detected a further enrichment for CD4+ over CD8+ cells (Fig. 1E). The continued proliferation of B and T cells in the triple mutants eventually filled their lymphoid compartments beyond capacity. Remarkably, we detected ectopic colonies of lymphocytes in every adult organ that we examined, including lung, liver, kidney, heart, pancreas, intestine, skeletal muscle, eye, brain, and spinal cord (Fig. 1, F and G).

The B and T cell populations of the triple mutants were also constituitively activated. Elevated numbers of triple mutant T cells, for example, expressed the interleukin-2 (IL-2) receptor (Fig. 2A) and the lectin CD69 (11), both markers of T cell activation. Similarly, triple mutant B cells displayed elevated surface expression of the acute activation marker Fas (Fig. 2B) and the chronic activation marker CD44 (Fig. 2C), and a pronounced increase in the expression of interferon-gamma (IFN-γ) was observed in the spleen and lymph nodes (Fig. 2D). The broad activation of B and T cells was, in turn, reflected in the activation of nonimmune tissues with which these cells interact. For example, intercellular adhesion molecule–1 (ICAM-1), which is required for lymphocyte adhesion to blood vessels and subsequent invasion of tissue parenchyma, was strongly up-regulated in the vascular endothelia of the triple mutants (Fig. 2E).

Figure 2

Activation of the immune system in the triple mutants. (A) Expression of the IL-2 receptor (CD25) on TCRαβ+ nodal T cells from wild-type and triple mutant individuals at 4 months of age. (B) Expression of Fas on B220+ splenic B cells from wild-type and triple mutant individuals at 2 months of age. (C) Expression of the chronic activation marker CD44 on B220+ lymph node B cells from wild-type and triple mutant individuals at 10 months of age. (D) In situ hybridization to sections of lymph nodes from 6-month-old micedemonstrating strong up-regulation of IFNγ mRNA in triple mutant (TAM) relative to wild type (WT). IFN-γ mRNA is abundantly expressed in T cell–rich regions of the triple mutant spleen (11). (E) Immunohistochemical staining of ICAM-1 on endothelial cells lining blood vessels in the brain (arrows) in triple mutants (TAM) relative to wild type (WT). Bars, 0.1 mm.

All triple mutants eventually developed autoimmunity. Disease symptoms, which were first detectable in a subset of individuals at ∼4 weeks after birth, were histologically similar to those seen in a broad spectrum of human autoimmune disorders, including rheumatoid arthritis (12) (Fig. 3A), pemphigus vulgaris (13) (Fig. 3B), and systemic lupus erythematosus (SLE) (14) (Fig. 3C). We detected recurrent thromboses and hemorrhage in several tissues, including the brain (Fig. 3D). These thromboses, which are associated with the presence of antibodies to phospholipids in human autoimmune syndromes (15), were especially prevalent in triple mutant females. Systemic autoimmune diseases result in elevated blood titers of antibodies directed against normal cellular antigens, including nucleoproteins and double-stranded (ds) DNA (16). Consistent with their autoimmune manifestations, we measured abnormally high levels of circulating antibodies to dsDNA throughout the allelic series of Tyro 3 family mutants (17,18) (Fig. 3E). In general, individuals carrying mutations in any two of the three genes exhibited higher α-dsDNA titers than did single mutants; on average, triple mutant titers were the highest (Fig. 3E). Also prevalent were autoantibodies to various collagens (Fig. 3F), which are frequently detected in the sera of patients with rheumatoid arthritis (12). Circulating antibodies to phospholipids, which are among the most reliable indicators of human autoimmune syndromes characterized by recurrent thromboses, hemolytic anemia, and, in women, chronic infertility due to recurrent fetal loss (19), appeared throughout the allelic series. We detected markedly elevated antibody titers to cardiolipin (Fig. 3G), phosphatidylserine (11), phosphatidylethanolamine (11), and phosphatidylinositol (Fig. 3H). Elevated α-cardiolipin titers were observed in several Tyro 3 andAxl single mutants, and individual triple mutants frequently displayed titers that were 20- to 40-fold higher than wild type (Fig. 3G). Most triple mutant females never carried pregnancies to term.

Figure 3

Clinical and humoral manifestations of autoimmunity in Tyro 3/ Axl/Mer triple mutants. (A) Swollen joints and footpad in a 6-month-old triple mutant female. Histologically, these symptoms were reflected in inflammation and lymphocyte invasion of the joints (11). (B) A typical skin lesion (arrowhead) on the upper back of an 8-month-old triple mutant female. (C) Immunoglobulin G (IgG) deposits in a glomerulus of the kidney (arrowhead) in a 12-month-old triple mutant female. (D) Blood vessel hemorrhages (arrowheads) in the brain of a 12-month-old triple mutant female. Bars, 0.1 mm. Circulating autoantibodies were measured in a 1:200 dilution of serum collected from single, double, and triple mutants of the Tyro 3 allelic series, against dsDNA (E), collagen (F), cardiolipin (G), and phosphatidylinositol (H). Points represent the average of triplicate determinations by solid-phase ELISA (17,18) in individual mice of the indicated genotypes. Genotype designations are as for Fig. 1C.

B and T cells do not express the three receptor genes that we inactivated. Previous work has established that Mer is expressed by peripheral blood and bone marrow mononuclear cells, monocytes, and macrophages, but not by granulocytes or peripheral blood B or T lymphocytes (6, 20). Similarly,Axl is expressed by CD34+ progenitor and bone marrow stromal cells and by peripheral monocytes and macrophages, but not by granulocytes or lymphocytes (21, 22). We used similar flow cytometric analyses with cell-specific markers to demonstrate that the Tyro 3 gene is also the product of monocytes and macrophages but not of B or T cells (11). In addition, we used in situ hybridization to examine the sites of expression of the Tyro 3, Axl, and MermRNAs, and of the Gas6 and Protein S mRNAs, in lymphoid tissues (Fig. 4, A through J). In the spleen, the mRNAs for all three receptors and both ligands were localized to the red pulp and to the marginal zones, and were largely excluded from the periarteriolar lymphoid sheath, the B cell corona, and the germinal centers of the white pulp, which contain the bulk of both B and T cells (circled signal-free areas in Fig. 4, A, C, E, G, and I). The Tyro 3 and Axl mRNAs were similarly absent from the primary lymphoid follicles and germinal centers of the lymph nodes (Fig. 4, B and D), which are predominantly composed of B cells, and the same was true for Gas6 and Protein S (Fig. 4, H and J). Each of these mRNAs were instead localized to paracortical and medullary cord regions of the lymph node, which contain macrophages and T cells. A similar pattern was observed forMer mRNA, although a small number of discreteMer + cells were also detected within lymphoid follicles (Fig. 4F). The Tyro 3, Axl, andMer mRNAs were each also confined to the central medulla of the thymus (23), a localization that excludes expression by immature T cells but is consistent with expression by dendritic cells and macrophages.

Figure 4

Expression of the Tyro 3 (Aand B), Axl (C and D), Mer (E and F), Gas 6 (G and H), and Protein S (I and J) mRNAs in lymphoid tissues. In situ hybridization was performed to sections of wild-type adult spleen (left column) and lymph node (right column). Dashed circles highlight signal-free areas in the white pulp of the spleen (left column) and lymphoid follicles of the lymph nodes (right column). Bar, 1 mm. See text for details. Host-dependent proliferation of wild-type B and T cells after transfer (K throughN). Dissociated spleen cells from wild-type females at 2 (M and N) and 2.5 (K and L) months of age were labeled with CFSE (24–26), and injected into the tail veins of wild-type (L and N) and triple mutant (K and M) females at 8 to 10 months of age. Cells were recovered from either the spleens (K and L) or lymph nodes (M and N) of the recipients at 4.5 (K and L) and 4 (M and N) days after injection; dissociated cells were then analyzed by flow cytometry for expression of the B cell marker B220 (K and L) or the T cell marker CD4 (M and N) and were simultaneously measured for CFSE fluorescence intensity (x axis, all panels). Dotted lines mark mean CFSE fluorescence peaks that correspond to absence of proliferation of the injected cells.

In addition to these expression analyses, we performed a series of in vivo transfer experiments in both wild-type and triple mutant mice. We labeled wild-type spleen cells with the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (24–26), which allowed us to measure the proliferation of the donor cells after injection. As measured by flow cytometry, successive rounds of cell division result in successive twofold diminutions in the fluorescence intensity of CFSE-labeled daughter cells. Three to 4 days after injection of CFSE-labeled cells, we analyzed spleens and lymph nodes of the injected mice for the number and fluorescence intensity of CFSE-labeled B220+ B cells and of labeled CD4+ and CD8+ T cells (Fig. 4, K through N). As expected, injection of wild-type cells into wild-type recipients (WT → WT) resulted in the recovery of nondividing B and T cell populations from the spleens and lymph nodes of the recipients (Fig. 4, L and N). In contrast, injection of the same cells into triple mutants (WT → TAM) resulted in the recovery of wild-type donor B and T cells that had undergone multiple rounds of cell division. The wild-type B cells that failed to proliferate in wild-type recipients were observed to undergo up to four rounds of cell division after 4.5 days in triple mutant spleens (Fig. 4K), and the wild-type T cells that failed to proliferate by 4 days after injection in wild-type recipients were observed to undergo up to three rounds of cell division by the same time in the triple mutant lymph nodes (Fig. 4M).

The above results suggest that rather than B and T lymphocytes, the cells that initiate the lymphoproliferation and autoimmunity of theTyro 3 family mutants are the macrophages and dendritic cells that normally express the three inactivated receptor genes. It has previously been noted that peritoneal macrophages cultured fromMer single mutants and challenged with bacterial lipopolysaccharide (LPS) express excessive levels of both activated nuclear factor kappa B (NF-κB) and the inflammatory cytokine tumor necrosis factor α (TNFα) (27, 28). Therefore, we examined the activation status of antigen-presenting cells (APCs) identified with the macrophage and dendritic cell markers CD11b and CD11c, at steady-state and in response to LPS stimulation, in triple mutants versus wild type (Fig. 5). Activated APCs express elevated levels of major histocompatibility complex (MHC) antigens on their surface, and this was the case for APCs in the triple mutants. Although MHC class II (I-Ab) levels were only modestly elevated in CD11c+ cells freshly dissociated from triple mutant spleen or lymph node (Fig. 5A, upper panels), these levels were superelevated in the same cells as an acute (1 hour) response to a 100 mg/kg intraperitoneal (IP) injection of LPS (Fig. 5A, lower panels). A similar LPS-induced superelevation of the B7.2 (CD86) co-receptor, also a marker of APC activation, was observed (Fig. 5B). Triple mutant peritoneal macrophages expressed aberrantly high MHC class II levels in cell culture, even in the absence of LPS stimulation (Fig. 5C, upper panels), and these levels were even further elevated 5 hours after exposure to LPS (Fig. 5C, lower panels). We also observed that cultured triple mutant macrophages produced excessive amounts of the proinflammatory cytokine IL-12 (Fig. 5D), and exhibited a 3.5 ± 0.9-fold increase (relative to wild type) in generalized phagocytosis in vitro, as assayed by the uptake of fluorescently labeled Escherichia coli after 1 hour of co-culture (28, 29). Finally, serum levels of TNFα were 11.3 ± 2.5 ng/ml 1 hour after injection of LPS into the triple mutants (n = 3 mice), as compared to 5.2 ± 1.1 ng/ml (n = 3) for wild type.

Figure 5

Hyperactivation of APCs in the triple mutants. (A) Expression of MHC class II protein on the surface of CD11c+ splenocytes isolated from wild-type (WT) and triple mutant (TAM) mice, before (–LPS) and 1 hour after (+LPS) an IP injection of LPS (100 mg/kg). (B) Expression of B7.2 on the surface of CD11c+splenocytes isolated from wild-type (WT) and triple mutant (TAM) mice, before (–LPS) and 1 hour after (+LPS) an IP injection of LPS. (C) Expression of MHC class II protein on the surface of wild-type (WT) and triple mutant (TAM) CD11b+ peritoneal macrophages in culture, before (–LPS) and 5 hours after (+LPS) exposure to LPS (1 μg/ml). (D) Expression of IL-12 in fixed and permeabilized wild-type (WT) and triple mutant (TAM) CD11b+ peritoneal macrophages in culture, before (–LPS) and 5 hours after (+LPS) exposure to LPS.

The salient features of the autoimmune phenotypes described above have important implications for the development of autoimmunity in humans (23). The chronic hyperactivation of APCs in the triple mutants indicates that signaling through the Tyro 3 family receptors normally serves as a self-extinguishing regulatory mechanism that limits the severity and time course of inflammatory immune responses. The frequently observed coexpression of the receptors and their ligands (9), together with the presence of conserved immunoreceptor tyrosine-based inhibition motifs (ITIM-like elements) (30) in the cytoplasmic domains of all three receptors, immediately suggest a molecular basis for this self-regulation (23), a model that we are currently testing.

  • * To whom correspondence should be addressed. E-mail: lemke{at}salk.edu

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