Ryotaro Yoshida

Leukocyte Integrin‐Dependent and Antibody‐Independent Cytotoxicity of Macrophage against Allografts

Macrophages (Mφs), but not T cells, infiltrating into the rejection site of either i.p. allografted Meth A (H‐2) fibrosarcoma cells in C57BL/6 (B6) (H‐2b) mice or BALB/c (H‐2d) skin onto B6 mice are cytotoxic against allografts with H‐2d specificity. To determine the mechanisms of specific killing of allografts by allograft‐induced Mφ (AIM), we raised ≈5,000 rat monoclonal antibodies (mAbs) against AIM and selected three of them (R1–73, R2–40 and R1–34), each of which inhibited cytotoxic activity against allografts in a dose‐dependent manner. The antigens recognized by R1–73, R2–40 and R1–34 mAbs were defined by immunoprecipitation and Western blot analyses as CD11a, CD18 and CD11b, respectively; and the allografts expressed CD54, a ligand of CD11a or CD11b, suggesting leukocyte integrin‐dependent killing. Although Ab‐dependent cellular cytotoxicity has been recognized as a mechanism of specific killing by Mφs, the infiltration of AIM into the rejection site of allografts far (≈6 days) preceded the appearance of serum IgG Ab specific for the allograft. AIM exhibiting full cytotoxic activity against allografts was also induced in the transplantation site of Fcγ receptor knockout [(B6×129) F1] mice as well as B10.D2 (H‐2 compatible with allograft) and B6‐xid (X‐linked immunodeficiency with B cell‐specific defect) strains of mice. In the latter two strains of mice, the levels of serum IgG Ab to the allograft were negligible. Moreover, the cytotoxic activity of AIM against allografts was not affected by pretreatment of the cells with anti‐mouse IgG serum, suggesting Ab‐independent cytotoxicity.

Differential Susceptibility of Cells Expressing Allogeneic MHC or Viral Antigen to Killing by Antigen-Specific CTL

CD8+ cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8+ T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus‐infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8‐knockout mice. To address this controversy, we generated H‐2d‐specific CD8+ CTLs by a mixed lymphocyte culture and examined the susceptibility of a panel of H‐2d cells to CTL lysis. KLN205 squamous cell carcinoma, Meth A fibrosarcoma, and BALB/c skin components were found to be resistant to CTL‐mediated lysis. This resistance did not appear to be related to a reduced expression of MHC class I molecules, and all these cells could block the recognition of H‐2d targets by CTLs in cold target inhibition assays. We extended our observation by persistently infecting the same panel of cell lines with defective‐interfering Sendai virus particles. The Meth A and KLN205 lines infected with a variant Sendai virus were resistant to lysis by Sendai virus‐specific CTLs. The Sendai virus‐infected Meth A and KLN205 lines were able to block the lysis of Sendai virus‐infected targets by CTLs in cold target inhibition assays. Taken together, these results suggest that not all in vivo tissues may be sensitive to CTL lysis.

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.

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).

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.

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.

HLA-B62 as a possible ligand for the human homologue of mouse macrophage MHC receptor 2 (MMR2) on monocytes.

We previously reported that a population of allograft (H-2D(d)K(d))-induced macrophages (AIM) in C57BL/6 (H-2D(b)K(b)) mice exhibited major histocompatibility complex (MHC) haplotype (H-2D(d) or H-2K(d))-specific killing of allografts in a macrophage MHC receptor 1 or 2 (MMR1 or 2)-dependent manner. In the present study, we isolated a cDNA encoding a human homologue (83.6% amino-acid identity) of mouse MMR2 from a human cDNA library, the donors of which had never been allografted. The cDNA (2376-bp) encoded a 791-amino-acid polypeptide with a calculated molecular mass of 91kDa. Unexpectedly, the mRNA was expressed at least in part in peripheral blood mononuclear cells (PBMCs) or monocytes, but not in granulocytes or lymphocytes. The expression varied from volunteer to volunteer: PBMCs from 8 volunteers expressed human MMR2 at similar levels, whereas those from 8 other volunteers showed no or much less expression of it. Flow cytometric analyses revealed that HEK293T cells expressing human MMR2 protein bound fluorescein-labeled HLA-B62, but not A2, A-11, A-24 or B7, with a dissociation constant (=8.9x10(-9)M) and that the interaction was completely inhibited by the addition of R12 mAb specific for mouse MMR2. Similarly, the expression of mouse MMR2 varied from strain to strain in mice: PBMCs from 9 non-H-2K(d), but not from 3 H-2K(d), mice expressed mouse MMR2 specific for H-2K(d). These results suggest that human MMR2 on monocytes may be a novel receptor for HLA-B62.

Down‐regulated expression of monocyte/macrophage major histocompatibility complex receptors in human and mouse monocytes by expression of their ligands

Mouse monocyte/macrophage major histocompatibility complex (MHC) receptor 1 (MMR1; or MMR2) specific for H‐2Dd (or H‐2Kd) molecules is expressed on monocytes from non‐H‐2Dd (or non‐H‐2Kd), but not those from H‐2Dd (or H‐2Kd), inbred mice. The MMR1 and/or MMR2 is essential for the rejection of H‐2Dd‐ and/or H‐2Kd‐transgenic mouse skin onto C57BL/6 (H‐2DbKb) mice. Recently, we found that human leucocyte antigen (HLA)‐B44 was the sole ligand of human MMR1 using microbeads that had been conjugated with 80 types of HLA class I molecules covering 94·2% (or 99·4%) and 92·4% (or 96·2%) of HLA‐A and B molecules of Native Americans (or Japanese), respectively. In the present study, we also explored the ligand specificity of human MMR2 using microbeads. Microbeads coated with HLA‐A32, HLA‐B13 or HLA‐B62 antigens bound specifically to human embryonic kidney (HEK)293T or EL‐4 cells expressing human MMR2 and to the solubilized MMR2‐green fluorescent protein (GFP) fusion protein; and MMR2+ monocytes from a volunteer bound HLA‐B62 molecules with a Kd of 8·7 × 10−9 M, implying a three times down‐regulation of MMR2 expression by the ligand expression. H‐2Kd (or H‐2Dd) transgene into C57BL/6 mice down‐regulated not only MMR2 (or MMR1) but also MMR1 (or MMR2) expression, leading to further down‐regulation of MMR expression. In fact, monocytes from two (i.e. MMR1+/MMR2+ and MMR1–/MMR2–) volunteers bound seven to nine types of microbeads among 80, indicating ≤ 10 types of MMR expression on monocytes.