Joan Stein-Streilein

Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages

In the inflamed cornea, there is a parallel outgrowth of blood and lymphatic vessels into the normally avascular cornea. We tested whether adaptive and/or innate immune cells were actively involved in the genesis of new lymphatic vessels. Our results indicate that innate immune cells (CD11b+ macrophages, but not CD11c+ dendritic cells) physically contributed to lymphangiogenesis under pathological conditions and that bone marrow-derived CD11b+ macrophages expressed lymphatic endothelial markers such as LYVE-1 and Prox-1 under inflamed conditions in the corneal stromata of mice. Furthermore, blood vascular endothelial cells that expressed the Tie2 promoter did not contribute to newly formed lymphatic vessels under inflamed conditions. Our in vitro experiments demonstrated that CD11b+ macrophages alone were capable of forming tube-like structures that expressed markers of lymphatic endothelium such as LYVE-1 and podoplanin. The novel finding that CD11b+ macrophages are critical for the development of inflammation-dependent lymphangiogenesis in the eye suggests a new mechanism of lymphangiogenesis.

The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance

We show that the mouse macrophage-restricted F4/80 protein is not required for the development and distribution of tissue macrophages but is involved in the generation of antigen-specific efferent regulatory T (T reg) cells that suppress antigen-specific immunity. In the in vivo anterior chamber (a.c.)–associated immune deviation (ACAID) model of peripheral tolerance, a.c. inoculation of antigen into F4/80−/− mice was unable to induce efferent T reg cells and suppress delayed-type hypersensitivity (DTH) responses. Moreover, the use of anti-F4/80 mAb and F4/80−/− APCs in an in vitro ACAID model showed that all APC cells in the culture must be able to express F4/80 protein if efferent T reg cells were to be generated. In a low-dose oral tolerance model, WT but not F4/80−/− mice generated an efferent CD8+ T reg cell population that suppressed an antigen-specific DTH response. Peripheral tolerance was restored in F4/80−/− mice by adoptive transfer of F4/80+ APCs in both peripheral tolerance models, indicating a central role for the F4/80 molecule in the generation of efferent CD8+ T reg cells.

NK T Cell-Derived IL-10 Is Essential for the Differentiation of Antigen-Specific T Regulatory Cells in Systemic Tolerance

In a model of systemic tolerance called Anterior Chamber-Associated Immune Deviation (ACAID), the differentiation of the T regulatory (Tr) cells depends on NK T cells and occurs in the spleen. We now show that the CD1d-reactive NK T cell subpopulation, required for development of systemic tolerance, expresses the invariant Vα14Jα281 TCR because Jα281 knockout (KO) mice were unable to generate Ag-specific Tr cells and ACAID. The mechanism for NK T cell-dependent differentiation of Ag-specific Tr cells mediating systemic tolerance was studied by defining the cytokine profiles in heterogeneous and enriched NK T spleen cells. In contrast to there being no differences in most regulatory cytokine mRNAs, both mRNA and protein for IL-10 were increased in splenic NK T cells of anterior chamber (a.c.)-inoculated mice. However, IL-10 mRNA was not increased in spleens after i.v. inoculation. Finally, NK T cells from wild-type (WT) mice, but not from IL-10 KO mice, reconstituted the ACAID inducing ability in Jα281 KO mice. Thus, NK T cell-derived IL-10 is critical for the generation of the Ag-specific Tr cells and systemic tolerance induced to eye-inoculated Ags.

MIP-2 Recruits NKT Cells to the Spleen During Tolerance Induction

Peripheral tolerance occurs after intraocular administration of Ag and is dependent on an increase in splenic NKT cells. New data here show that macrophage inflammatory protein-2 (MIP-2) is selectively up-regulated in tolerance-conferring APCs and serves to recruit NKT cells to the splenic marginal zone, where they form clusters with APCs and T cells. In the absence of the high-affinity receptor for MIP-2 (as in CXCR2-deficient mice) or in the presence of a blocking Ab to MIP-2, peripheral tolerance is prevented, and Ag-specific T regulatory cells are not generated. Understanding the regulation of lymphocyte traffic during tolerance induction may lead to novel therapies for autoimmunity, graft acceptance, and tumor rejection.

ANTERIOR CHAMBER ASSOCIATED IMMUNE DEVIATION (ACAID): REGULATION, BIOLOGICAL RELEVANCE, AND IMPLICATIONS FOR THERAPY

Immune privilege was first explored in the late 1800s by van Dooremaal, and was then extended by Medawar in the mid 1900s to fit in with emerging concepts of transplantation immunology. Modern concepts and understanding of immune privilege come from subsequent studies produced by Medawar, Billingham, and Streilein. The exploitation of the model of anterior chamber immune deviation (ACAID) in mice has allowed us to look at both cellular and molecular mechanisms involved in the prevention of potentially damaging immune responses in such privileged sites. This review gives a historical perspective of the immune privilege research and provides up-to-date information of molecules, cells, and concepts newly recognized as contributing to tolerance induction induced in such specialized areas of the body. Evidence is given to support the idea that application of such information may lead to potential for therapeutic applications of ACAID mechanisms in prevention of progression of immune-inflammatory diseases in humans.

Cutting Edge: In Vitro-Generated Tolerogenic APC Induce CD8+ T Regulatory Cells That Can Suppress Ongoing Experimental Autoimmune Encephalomyelitis

APC exposed to TGFβ2 and Ag (tolerogenic APC) promote peripheral Ag-specific tolerance via the induction of CD8+ T regulatory cells capable of suppressing Th1 and Th2 immunity. We postulated that tolerogenic APC might reinstate tolerance toward self-neuronal Ags and ameliorate ongoing experimental autoimmune encephalomyelitis (EAE). Seven days after immunization with myelin basic protein (MBP), mice received MBP-specific tolerogenic APC, and EAE was evaluated clinically. To test for the presence and the phenotype of T regulatory cells, CD4 and/or CD8 T cells from tolerogenic APC-treated mice were transferred to naive mice before their immunization with MBP. The MBP-specific tolerogenic APC decreased both the severity and incidence of ongoing EAE. Tolerance to self-neuronal Ags was induced in naive recipient mice via adoptive transfer of CD8+, but not CD4+ T cells. Rational use of in vitro-generated tolerogenic APC may lead to novel therapy for autoimmune disease.

NKT Cell-Derived RANTES Recruits APCs and CD8+ T Cells to the Spleen During the Generation of Regulatory T Cells in Tolerance

The induction of peripheral tolerance via immune privileged sites such as the eye requires splenic colocalization of NKT cells and CD1d+ tolerogenic F4/80+ APCs, both of which are needed for the generation of CD8+-regulatory T (Tr) cells. Whereas tolerogenic APCs secrete the chemokine macrophage-inflammatory protein-2 for the purpose of recruiting NKT cells, the signals responsible for recruiting potential Tr cells and additional APCs to the spleen are not known. Here we examined the ability of CD1d-stimulated NKT cells to produce chemokines that can recruit other cells needed for tolerance. Our results show that NKT cells stimulated by either CD1d-transfected fibroblasts in vitro or CD1d+ tolerogenic APCs both in vivo and ex vivo produced RANTES in a CD1d-dependent manner. The requirement for RANTES in tolerance was demonstrated by studies in which RANTES blockade in vivo prevented not only APC accumulation in the spleen but also the generation of CD8+ Tr cells that suppress Th1 immunity. Thus, CD1d-restricted NKT cells provide critical signals for orchestrating the accumulation of cells needed for tolerance induction. These data expand our current knowledge of RANTES beyond its role in Th1 immune responses to show its importance in tolerance induction and add a novel aspect to our understanding of the role of NKT cells in tolerance. Understanding the precise mechanisms involved in tolerance induction may lead to more effective therapeutic strategies for autoimmunity and graft rejection.

CD4+ NKT Cells, But Not Conventional CD4+ T Cells, Are Required to Generate Efferent CD8+ T Regulatory Cells Following Antigen Inoculation in an Immune-Privileged Site

Following inoculation of Ag into the anterior chamber (a.c.), systemic tolerance develops that is mediated in part by Ag-specific efferent CD8+ T regulatory (Tr) cells. This model of tolerance is called a.c.-associated immune deviation. The generation of the efferent CD8+ Tr cell in a.c.-associated immune deviation is dependent on IL-10-producing, CD1d-restricted, invariant Vα14+ NKT (iNKT) cells. The iNKT cell subpopulations are either CD4+ or CD4−CD8− double negative. This report identifies the subpopulation of iNKT cells that is important for induction of the efferent Tr cell. Because MHC class II−/− (class II−/−) mice generate efferent Tr cells following a.c. inoculation, we conclude that conventional CD4+ T cells are not needed for the development of efferent CD8+ T cells. Furthermore, Ab depletion of CD4+ cells in both wild-type mice (remove both conventional and CD4+ NKT cells) and class II−/− mice (remove CD4+ NKT cells) abrogated the generation of Tr cells. We conclude that CD4+ NKT cells, but not the class II molecule or conventional CD4+ T cells, are required for generation of efferent CD8+ Tr cells following Ag introduction into the eye. Understanding the mechanisms that lead to the generation of efferent CD8+ Tr cells may lead to novel immunotherapy for immune inflammatory diseases.

Long-Term Survival of Corneal Allografts Is Dependent on Intact CD1d-Reactive NKT Cells

BALB/c mice that tolerate the allogeneic grafts develop allogeneic-specific anterior chamber-associated immune deviation. Because CD1d-reactive NKT cells are essential for anterior chamber-associated immune deviation, we postulated that the survival of C57BL/6 (B6) cornea graft in BALB/c mice was also dependent on CD1d-reactive NKT cells. The B6 corneal graft rejection rate in BALB/c vs Jα281 knockout (KO) mice, which lack NKT cells, was measured. While there were no difference in the early phase of rejection, the survival rates at 12 wk after grafting for BALB/c and Jα281 KO mice were 50 and 0%, respectively. Because anti-CD1d mAb abrogated the corneal graft survival in the wild-type mice we concluded that CD1d-reactive NKT cells were essential for graft survival. Moreover, allospecific T regulatory (Tr) cells correlated with acceptance of B6 grafts in BALB/c mice, and the adoptive transfer of these allospecific Tr cells to Jα281 KO mice allowed a 50% survival rate of B6 cornea grafts. In conclusion, CD1d-reactive NKT cells are required for induction of allospecific Tr cells and are essential for survival of corneal allografts. Mechanisms that contribute to cornea graft acceptance may lead to new therapies for improvement in graft survival in high-risk corneas and other transplanted tissues and grafts.

Does innate immune privilege exist

Immune privilege in the eye is believed to originate from the eyes need to avoid the sight‐destroying consequences of inflammation. Over the past 25 years, many of the anatomical, cellular, and molecular mechanisms by which the eye avoids inflammation secondary to adaptive immune responses have been elucidated. In the recent past, it has become increasingly clear that innate immune responses play a critical role in activating the adaptive immune response. Moreover, innate immunity itself carries a heavy burden of inflammation, thereby posing a threat to vision if it should occur intraocularly. Ocular immunologists have now begun to inquire into the extent to which the eye regulates the expression of innate immunity in oculi. Evidence is presented which indicates that factors found in normal aqueous humor (1) prevent NK cells from lysing their targets, (2) inhibit neutrophil activation by CD95 ligand, (3) suppress nitric oxide production by activated macrophages, and (4) interfere with complement activation via the alternative pathway. These factors include transforming growth factor‐β2, α‐melanocyte‐stimulating hormone, calcitonin gene‐related peptide, and migration inhibitory factor. The ability of the eye to prevent intraocular activation of innate immune effectors spares the corneal endothelium (which expresses CD95 ligand constitutively, but low levels of MHC class I molecules) from destruction by NK cells and neutrophils, and protects the visual axis from distortion by macrophage and complement‐mediated inflammation. Thus, privilege exists in the eye for both adaptive and innate immunity. J. Leukoc. Biol. 67: 479–487; 2000.