Junko Tashiro-Yamaji

Acute Rejection of Allografted CTL-Susceptible Leukemia Cells from Perforin/Fas Ligand Double-Deficient Mice

The generation of knockout mice demonstrated that CD4+, but not CD8+, T cells were essential for the rejection of allografted skin or heart, presumably because these targets were CTL resistant. In the case of CTL-susceptible targets (e.g., P815 mastocytoma cells and EL-4 or RLmale1 T lymphoma cells), however, it is assumed that the CTL is the effector cell responsible for allograft rejection and that perforin and Fas ligand (FasL) pathways are the killing mechanisms. In the present study, we examined the role of these cytotoxic molecules in the rejection of i.p. allografted CTL-susceptible leukemia cells. Unexpectedly, the allografted leukemia cells were acutely rejected from gld (a mutation of FasL), perforin−/−, or double-deficient mice. The peritoneal exudate cells from gld or normal mice showed T cell-, TCRαβ-, and perforin-dependent cytotoxic activity against the allograft, whereas the exudate cells from perforin−/− mice exhibited almost full cytotoxic activity in the presence of Fas-Fc. Furthermore, the infiltrates from double-deficient mice showed a high cytotoxic activity against the allografted cells even in the presence of anti-TCRαβ Ab or in the absence of T cells. The cytotoxic cells appeared to be macrophages, because they were Mac-1+ mononuclear cells with a kidney- or horseshoe-shaped nucleus and because the cytotoxic activity was completely suppressed by the addition of NG-monomethyl-l-arginine, an inhibitor of inducible NO synthase. These results indicate that macrophages are ready and available to kill CTL-susceptible allografts when CTLs lack both perforin and FasL molecules.

A Novel Receptor on Allograft (H-2d)-Induced Macrophage (H-2b) toward an Allogeneic Major Histocompatibility Complex Class I Molecule, H-2Dd, in Mice

The generation of knockout mice demonstrated that noncytotoxic CD4+, but not cytotoxic CD8+, T cells were essential for the rejection of skin or organ allografts. Earlier we reported that allograft‐induced macrophages (AIM) in mice lysed allografts with H‐2 haplotype specificity, implying screening of grafts by AIM. Here, we isolated a cDNA clone encoding a novel receptor on AIM (H‐2Db) for an allogeneic major histocompatibility complex (MHC) class I molecule, H‐2Dd, by using H‐2Dd tetramer and a monoclonal antibody (mAb; R15) specific for AIM. The cDNA (1,181‐bp) encoded a 342‐amino acid polypeptide with a calculated molecular mass of 45 kDa and was found to be expressed on AIM, but not on resident macrophages or other cells, infiltrating into the rejection site. HEK293T cells transfected with this cDNA reacted with R15 mAb and H‐2Dd, but not H‐2Ld, H‐2Kd, H‐2Db, H‐2Kb, H‐2Dk, or H‐2Kk, molecules; and the H‐2Dd binding was suppressed by the addition of R15 or anti‐H‐2Dd mAb. AIM yielded a specific saturation isotherm in the presence of increasing concentrations of H‐2Dd, but not H‐2Db or H‐2Dk, molecules. The dissociation constant of AIM toward H‐2Dd tetramers was 1.9×10–9 M; and the binding was completely inhibited by the addition of R15 or anti‐H‐2Dd mAb. These results reveal that a novel receptor for an allogeneic H‐2Dd molecule was induced on effector macrophages responsible for allograft (H‐2d) rejection in H‐2b mice.

Rejection of intradermally injected syngeneic tumor cells from mice by specific elimination of tumor-associated macrophages with liposome-encapsulated dichloromethylene diphosphonate, followed by induction of CD11b+/CCR3−/Gr-1− cells cytotoxic against the tumor cells

Tumor cell expansion relies on nutrient supply, and oxygen limitation is central in controlling neovascularization and tumor spread. Monocytes infiltrate into tumors from the circulation along defined chemotactic gradients, differentiate into tumor-associated macrophages (TAMs), and then accumulate in the hypoxic areas. Elevated TAM density in some regions or overall TAM numbers are correlated with increased tumor angiogenesis and a reduced host survival in the case of various types of tumors. To evaluate the role of TAMs in tumor growth, we here specifically eliminated TAMs by in vivo application of dichloromethylene diphosphonate (DMDP)-containing liposomes to mice bearing various types of tumors (e.g., B16 melanoma, KLN205 squamous cell carcinoma, and 3LL Lewis lung cancer), all of which grew in the dermis of syngeneic mouse skin. When DMDP-liposomes were injected into four spots to surround the tumor on day 0 or 5 after tumor injection and every third day thereafter, both the induction of TAMs and the tumor growth were suppressed in a dose-dependent and injection number-dependent manner; and unexpectedly, the tumor cells were rejected by 12 injections of three times-diluted DMDP-liposomes. The absence of TAMs in turn induced the invasion of inflammatory cells into or around the tumors; and the major population of effector cells cytotoxic against the target tumor cells were CD11b+ monocytic macrophages, but not CCR3+ eosinophils or Gr-1+ neutrophils. These results indicate that both the absence of TAMs and invasion of CD11b+ monocytic macrophages resulted in the tumor rejection.

Infiltration of H-2d-specific cytotoxic macrophage with unique morphology into rejection site of allografted meth A (H-2d) tumor cells in C57BL/6 (H-2b) mice

It is assumed that CD8+ cytotoxic T lymphocytes (CTLs) mediate direct lysis of allografts and that their growth, differentiation, and activation are dependent upon cytokine production by CD4+ helper T lymphocytes. In the present study, the effector cells responsible for the rejection of i.p. allografted, CTL‐resistant Meth A tumor cells from C57BL/6 mice were characterized. The cytotoxic activity was associated exclusively with peritoneal exudate cells and not with the cells in lymphoid organs or blood. On day 8, when the cytotoxic activity reached a peak, 3 types of cells (i.e., lymphocytes, granulocytes, and macrophages) infiltrated into the rejection site; and allograft‐induced macrophages (AIM) were cytotoxic against the allograft Bacterially‐elicited macrophages also exhibited cytotoxic activity (≈1/2 of that of AIM) against Meth A cells, whereas the cytotoxic activity of AIM against these cells but not that of bacterially‐elicited macrophages was completely inhibited by the addition of donor (H‐2d)‐type lymphoblasts, suggesting H‐2d‐specific cytotoxicity of AIM against Meth A cells. In contrast, resident macrophages were inactive toward Meth A cells. Morphologically, the three‐dimensional appearance of AIM showed them to be unique large elongated cells having radiating peripheral filopodia and long cord‐like extensions arising from their cytoplasmic surfaces. The ultrastructural examination of AIM revealed free ribosomes in their cytoplasm, which was often deformed by numerous large digestive vacuoles. These results indicate that AIM are the H‐2d‐specific effector cells for allografted Meth A cells and are a more fully activated macrophage with unique morphological features.

Experimental autoimmune uveoretinitis initiated by non-phagocytic destruction of inner segments of photoreceptor cells by Mac-1+ mononuclear cells

EAU in mice is a model of human posterior uveitis. EAU is a Th1‐dependent disease that has been assumed to target the neural retina and related tissues; however, in situ effector cells and the target have not yet been clearly demonstrated. In the present study, we induced EAU in B10R mice by immunizing them with human interphotoreceptor retinoid‐binding protein peptide 161–180. Histological examinations revealed that EAU occurred approximately 11 days after the immunization and reached a peak on day 14. Retinae from normal or EAU mice were treated with proteases to obtain mono‐dispersed cells. The mono‐dispersed cells thus obtained were separated into three to four fractions by discontinuous Percoll density‐gradient (e.g. PBS/40/60) centrifugation. In normal mice, 94% of the total cells were recovered in two fractions (i.e. PBS/40 and pellet); and these fractions mainly contained inner and outer segments and cell bodies of photoreceptor cells and RPE cells, respectively. In EAU mice, additional cells (i.e. inflammatory cells) were obtained at the 40/60 interface. Electron microscopic examination showed that tissue damage during EAU was initiated by non‐phagocytic destruction of inner segments by Mac‐1+ mononuclear cells on day 11, followed by phagocytic activity of macrophages against outer segments and RPE cells on day 14. In vitro culturing of normal retinal cells with EAU infiltrates suggested the involvement of TNF‐α and NO in the tissue damage. These results indicate that EAU was initiated by non‐phagocytic destruction of inner segments of photoreceptor cells by Mac‐1+ mononuclear cells.

Two types of allograft-induced cytotoxic macrophage, one against allografts and the other against syngeneic or allogeneic tumor cells

In the 1990s, based on the results of studies using β2M, CD4 or CD8 knockout mice, several groups reported that the main effector cells responsible for skin or organ allograft rejection were non‐T, non‐NK cells. Similarly, we demonstrated that in an animal model of transplantation of BALB/c (H‐2d) skin onto or Meth A (H‐2d) tumor cells into C57BL/6 (H‐2b) mice, AIM, which expressed iNOS, IL‐12, and IL‐18, were the main effector cells and also that they were cytotoxic against syngeneic tumor cells. Here, we examined whether the same population of macrophages could react with two distinct types of target cell. When BALB/c skin or Meth A tumor cells were transplanted into C57BL/6 mice, cytotoxic activity against the allograft was induced in the transplantation site on days 5–14 and was recovered in non‐adherent cells after a 20‐min incubation in a serum‐coated dish, suggesting the induction of a type of AIM (AIM‐1) in the transplantation site. The AIM‐1‐expressing receptors for H‐2DdKd antigens had no cytotoxic activity against syngeneic tumor cells. In contrast, AIM‐2, which were recovered in the fraction adherent to the serum‐coated dish, exhibited cytotoxic activities against various types of tumor cells, whereas they were inactive toward BALB/c skin. AIM expressed iNOS (AIM‐1 < AIM‐2), IL‐12 (AIM‐1 > AIM‐2), and IL‐18 (AIM‐2 alone) mRNAs. These results indicate that after allografting, two distinct types of cytotoxic AIM were induced in the transplantation site, one against the allografted skin or tumor (AIM‐1) and the other against allogeneic or syngeneic tumor cells (AIM‐2).

Essential Role of Monocytes in the In Vitro Production of IL-4 and Nonspecific IgE Antibody by Peripheral Blood Lymphocytes from Mice Sensitized s.c. Once with Cedar Pollen

To explore which cytokine or cell is essential for the production of antibodies (Abs) of the IgE class in allergic diseases, we injected cedar pollen into wild-type, interferon-gamma(-/-) (IFN-gamma(/)), or interleukin-4(-/-) (IL-4(-/-)) BALB/c mice through four (i.n., i.p., s.c., and i.v.) different routes without adjuvant. Wild-type or IFN-gamma(-/-), but not IL-4(-/-), mice sensitized once or twice showed a significant increase in total IgE Ab in their serum, revealing the essential role of IL-4 in the production of total IgE Ab. We separated peripheral blood mononuclear cells (PBMCs) from untreated or sensitized mice into monocyte-rich, lymphocyte-rich, and granulocyterich populations by Percoll density-gradient centrifugation or into specific antigen cells by flow cytometry, cultured the cells in various combinations, and examined the levels of cytokines and IgE Ab released into the medium. The PBMCs from mice sensitized s.c. once, but not those from untreated animals, produced significant amounts of IL-4 and total IgE Ab, whereas the lymphocyte-rich population alone did not. Unexpectedly, IL-4 and IgE Ab production was restored by the addition of Mac-1(+) cells in the monocyte-rich fraction to the lymphocyte-rich fraction. These results indicate the essential role of monocytes in the production of IL-4 and total IgE Ab by lymphocytes during the initial stage of sensitization.

IgE Production after Four Routes of Injections of Japanese Cedar Pollen Allergen without Adjuvant: Crucial Role of Resident Cells at Intraperitoneal or Intranasal Injection Site in the Production of Specific IgE toward the Allergen

The production of specific IgE antibodies directed toward cedar pollen correlates well with the onset of allergic rhinitis; but the mechanisms of allergen recognition as nonself and Ig class switch to IgE by the immune system are still not fully understood. In the present study, we injected cedar pollen into mice through 4 different routes (intranasal (i.n.), intraperitoneal (i.p.), intravenous (i.v.), and subcutaneous (s.c.)) without adjuvant 1 to 3 times, and determined time‐dependent changes in the total and specific serum IgE levels compared with those in the serum levels of other isotype Igs. After an i.p. or i.n. injection of allergen into the mice, they produced a 1.5‐ to 1.7‐fold increase in total IgE, but none in IgG, IgM, or IgA antibodies in their serum, whereas an i.v. or s.c. injection of allergen was inactive as an inducer of total IgE antibodies. Upon a 2nd (s.c.) injection of the allergen into the i.p. or i.n. sensitized mice, a large amount of allergen‐specific IgE antibodies was found in the serum. In the case of i.v. or s.c. sensitized mice, however, they produced total, but not specific, IgE antibodies; and a 3rd (s.c.) injection of the allergen resulted in a large amount of specific IgE antibodies in the serum. These results imply that resident cells at the i.p. or i.n. injection site may play a crucial role in the efficient production of total and specific IgE antibodies toward the allergen.

Macrophage MHC and T-cell receptors essential for rejection of allografted skin and lymphoma.

Background Skin or organ allograft rejection is dependent on noncytotoxic CD4+ T cells, but the mechanisms of recognition and rejection remain elusive. Previously, we demonstrated C57BL/6 (H-2DbKb) macrophage-mediated, cell-to-cell contact-dependent, d haplotype-specific lysis of allografts (e.g., BALB/c skin and Meth A cells; H-2DdKd) in the rejection site and isolated two cDNA clones encoding receptors on macrophages for H-2Dd and H-2Kd, macrophage major histocompatibility complex receptor (MMR) 1 and 2, respectively. Methods To elucidate the role of MMR2 and T-cell receptors (TCRs) in graft rejection, we generated MMR2 knockout (KO) mice on a C57BL/6 background and transplanted Dd, Kd, or DdKd transgenic C57BL/6 skin or EL-4 lymphoma cells onto or into these KO mice. Results MMR2 KO mice lacking MMR2 mRNA or protein expression in their monocytes had no obvious abnormalities in terms of cell number in or composition of their lymphoid tissues or in T lymphocyte responses to alloantigen or nonalloantigen, whereas they failed to reject Kd transgenic skin grafts. Surprisingly, they also lacked MMR1 mRNA and protein expression in their monocytes and failed to reject Dd or DdKd transgenic skin grafts. However, they did reject skin grafts from mice expressing H-2Id, minor Hd, or third-party major histocompatibility complex. On the contrary, Dd-, Kd-, or DdKd-EL-4 cells injected intradermally or intraperitoneally into MMR2 KO mice were rejected by TCR&agr;&bgr;+/CD8+ T cells in a transgene number-dependent and MMR-independent manner. Conclusions These results demonstrate that MMRs on monocytes/macrophages and TCRs on cytotoxic T lymphocytes in mice were essential for recognition and rejection of allografted skin and lymphoma, respectively.

Specific binding of HLA-B44 to human macrophage MHC receptor 1 on monocytes.

Allograft (H-2D(d)K(d))-induced macrophages (AIM) in C57BL/6 (H-2D(b)K(b)) mice exhibit major histocompatibility complex (MHC) haplotype-specific killing of allografts in a macrophage MHC receptor 1 (MMR1; for H-2D(d))- and MMR2 (for H-2K(d))-dependent manner. Recently, we showed HLA-B62 to be a ligand for the human homologue of mouse MMR2. In the present study, we isolated a cDNA encoding the human homologue of mouse MMR1 and found HLA-B44 to be the sole ligand specific for the human MMR1 by using beads that had been conjugated with 80 kinds of HLA proteins. Flow cytometric analyses revealed that HLA-B44-conjugated beads are specifically bound to HEK293T cells expressing human MMR1, that HLA-B44 tetramers are bound to the human MMR1-transfected HEK293T cells with a dissociation constant of 3.0×10(-9) M, and that the interaction was completely inhibited by the addition of R15 monoclonal antibody specific for mouse MMR1. The MMR1 cDNA (1537-bp) encoded a 473-amino acid polypeptide and was expressed at least in part in the brain and peripheral blood mononuclear cells (PBMCs) or monocytes, but not in granulocytes or lymphocytes. PBMCs from 7 non-H-2D(d) (non-self), but none from 5 H-2D(d) (self), in-bred mice expressed mouse MMR1 specific for H-2D(d). In contrast, PBMCs from none of the 16 human volunteers expressed HLA-B44; whereas those from only 3 of these 16 volunteers expressed human MMR1. These results reveal that human MMR1 on monocytes is a novel receptor specific for HLA-B44.