Role of cytokines in lavage or drainage fluid after hemithyroidectomy in wound healing: involvement of histamine in the acceleration and delay of wound healing.

Wound healing is a sophisticated biologic process. In the case of hemithyroidectomy, the operation time is relatively short with small tissue damage and without skin excision, and bacterial contamination before, during, and after the operation is uncommon. Here, we explored which cytokine(s) affected the rates of healing of skin wounds after hemithyroidectomy of 29 patients. We assessed the amounts of cytokines (e.g., interleukin-6, platelet-derived growth factor, basic fibroblast growth factor, vascular endothelial growth factor, and tumor necrosis factor-α) in either the preoperative or postoperative lavage fluids, or in the drainage fluids on postoperative days (PODs) 1-8. All of these cytokines showed a similar pattern; after reaching a peak on POD1, the production fell sharply on POD2-8, revealing that wound healing commenced on POD1. The rates of wound healing were inversely related to the levels of histamine in six patients (i.e., those with the three largest and those with the three smallest total volumes of drainage fluid on POD1): high (or low) levels of histamine in the postoperative lavage fluids with low (or high) levels in the drainage fluids on POD1 caused earlier (or the delay of) wound healing, suggesting involvement of histamine in the acceleration and delay of wound healing.

Essential role of macrophages in the initiation of allergic rhinitis in mice sensitized intranasally once with cedar pollen: regulation of class switching of immunoglobulin in B cells by controlling interleukin-4 production in T cells of submandibular lymph nodes.

The production of allergen-specific IgE antibodies (Abs) in allergen-sensitized patients or animals has a mutual relationship with the immunologic response leading to allergic rhinitis. We recently reported that, after an intranasal injection of cedar pollen into mice, an interleukin-4 (IL-4)-dependent increase in serum nonspecific IgE Abs was a prerequisite for the production of serum allergen-specific IgE Abs. Here, we explored which lymphoid organs were responsive to the intranasally injected allergen and how IL-4 and IgE Abs were produced in the lymphocytes. Time-dependent changes in the total cell numbers and in in vitro IgE Ab production in various lymphoid organs revealed that the submandibular lymph nodes were the main responsible organ. After treatment with allergen (for IgE production) or allergen and complete Freund's adjuvant (for IgG production), we separated submandibular lymph node cells into macrophage-, lymphocyte-, and granulocyte-rich populations by discontinuous Percoll density-gradient centrifugation. Unexpectedly, bulk cells, but not the lymphocyte- or macrophage-rich populations, produced significant amounts of IL-4, IgE, and IgG; whereas production was restored by addition of Mac-1(+) cells from the macrophage-rich to the lymphocyte-rich fraction. Furthermore, a combination of the lymphocyte-rich population (for IgG [or IgE]) production) and the macrophage-rich population (for IgE [or IgG]) production) produced a large amount of IgE (or IgG). These results indicate that, in the initiation of allergic rhinitis, macrophages in the submandibular lymph nodes are essential not only for IL-4 or immunoglobulin production, but also for class switching of immunoglobulin in lymphocytes.

Transgene number-dependent, gene expression rate-independent rejection of Dd -, Kd-, or Dd Kd -transgened mouse skin or tumor cells from C57BL/6 Db Kb mice.

Recipient cells migrating into the transplantation site of an allograft recognize histocompatibility antigens on the grafts and are cytotoxic against the grafts. Although the alloreactive immune response is predominantly directed at the major histocompatibility complex (major histocompatibility complex [MHC]; H-2 in mice) class I molecules, the basic mechanisms of allograft rejection (e.g., ligand-receptor interaction) remain unclear, because of the polymorphism and complexity of the MHC. To examine the role of MHC class I molecules in allograft rejection, D(d) , K(d) or D(d) K(d) -transgenic skin or tumor cells we established on a C57BL/6 (D(b) K(b) ) background and transplanted into C57BL/6 mice. Skin grafts from allogeneic (i.e., BALB/c, B10.D2, and BDF1) strains of mice were rejected from C57BL/6 mice on days 12-14 after grafting, whereas isografts were tolerated by these mice. Unexpectedly, skin grafts from D(d) -, K(d) -, and D(d) K(d) -transgenic C57BL/6 mice were rejected on days 12-14 in a transgene expression rate-independent manner from 9/19 (47%), 20/39 (51%), and 12/17 (71%) of C57BL/6 mice, respectively. Similarly, intradermally transplanted allogeneic (i.e., Meth A), but not syngeneic (i.e., EL-4), tumor cells were rejected from C57BL/6 mice; the growth of D(d) - or K(d) -transfected EL-4 cells was delayed by 10-13 days; and 4/10 (40%) of D(d) K(d) -transfected tumor cells were rejected from C57BL/6 mice. These results indicate that D(d) and K(d) genes are equivalent as allogeneic MHC class I genes and that C57BL/6 (D(b) K(b) ) mice reject D(d) -, K(d) -, or D(d) K(d) -transgened skin or tumor cells in a transgene number-dependent, gene expression rate-independent manner.

Spontaneous rejection of intradermally transplanted non-engineered tumor cells by neutrophils and macrophages from syngeneic strains of mice.

It is not surprising that tumors arising spontaneously are rarely rejected by T cells, because in general they lack molecules to elicit a primary T-cell response. In fact, cytokine-engineered tumors can induce granulocyte infiltration leading to tumor rejection. In the present study, we i.d. injected seven kinds of non-engineered tumor cells into syngeneic strains of mice. Three of them (i.e. B16, KLN205, and 3LL cells) continued to grow, whereas four of them (i.e. Meth A, I-10, CL-S1, and FM3A cells) were spontaneously rejected after transient growth or without growth. In contrast to the i.d. injection of B16 cells into C57BL/6 mice, which induces infiltration of TAMs into the tumors, the i.d. injection of Meth A cells into BALB/c mice induced the invasion of cytotoxic inflammatory cells, but not of TAMs, into or around the tumors leading to an IFN-γ-dependent rejection. On day 5, the cytotoxic activity against the tumor cells reached a peak; and the effector cells were found to be neutrophils and macrophages. The i.d. Meth A or I-10 cell-immunized, but not non-immunized, mice rejected i.p.- or i.m.-transplanted Meth A or I-10 cells without growth, respectively. The main effector cells were CTLs; and there was no cross-sensitization between these two kinds of tumor cells, suggesting specific rejection of tumor cells by CTLs from i.d. immunized mice. These results indicate that infiltration of cytotoxic myeloid cells (i.e. neutrophils and macrophages, but not TAMs) into or around tumors is essential for their IFN-γ-dependent spontaneous rejection.

IFN-γ Control of an Effector/Target Combination for Skin Allograft Rejection: Macrophage/Skin Components in Normal Mice or T Cell/Endothelial Cells in IFN-γ-Deficient Mice.

Organ, skin, or cell allografts are acutely rejected from normal mice, whereas vascularized organ allografts, but not allografted Meth A cells, are rejected from interferon-γ (IFN-γ)-deficient mice. Here we explored effector/target combinations for i.p. allografted Meth A (cytotoxic T lymphocyte [CTL]-resistant) or RLmale1 (CTL-susceptible) cells into or for BALB/c skin (skin components: CTL resistant) onto normal or IFN-γ-deficient C57BL/6 mice. After allografting, normal mice showed more infiltration but only a little thrombosis/hemorrhage. Monocyte/macrophage MHC receptor (MMR)+ macrophages (on days 5-10) and T cell receptor (TCR)+ CTLs (on days 7-9) were cytotoxic against Meth A cells or skin components and RLmale1 cells, respectively, and the allografts were rejected. After allografting into IFN-γ-deficient mice, MMR- macrophages and highly activated TCR+ CTLs were induced, and the mice died of hemorrhagic ascites with Meth A cells and more acutely rejected RLmale1 cells. The CTLs on days 4-6 were inactive toward skin components at an in vivo effector/target ratio but injured endothelial cells to cause severe thrombosis/hemorrhage and more acute rejection of skin allografts. These results indicate that IFN-γ-dependent MMR expression was essential for macrophage-mediated cytolysis of allogeneic skin components and that IFN-γ-deficient mice more acutely rejected skin allograft by causing CTL-induced injury to endothelial cells.

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.

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.

Macrophage MHC receptor 2: a novel receptor on allograft (H-2D(d)K(d))-induced macrophage (H-2D(b)K(b)) recognizing an MHC class I molecule, H-2K(d), in mice.

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)) specific killing of the allograft (e.g., BALB/c skin and Meth A cells; H-2D(d)K(d)) in a macrophage MHC receptor (MMR)-dependent manner. In the present study, we isolated a cDNA clone encoding a novel receptor (MMR2) on AIM recognizing another MHC class I molecule, H-2K(d), by the expression cloning method using H-2K(d) tetramer and a monoclonal antibody (mAb; R12) specific for AIM. The cDNA (2359-bp) encoded a 677-amino acid polypeptide of a calculated molecular mass of 87 kDa and was found to be expressed exclusively on AIM among cells infiltrating into allografts on days 0-9 after transplantation. Confocal microscopy showed that HEK293T cells transfected with this cDNA were reactive toward the H-2K(d) molecule but not toward other MHC class I molecules such as H-2D(d), H-2D(b), H-2D(k), H-2K(b), H-2K(k), and H-2L(d) molecules. The binding of the H-2K(d) molecule to the transfectants was inhibited by the addition of R12 or anti-H-2K(d), but not by R15 (a mAb specific for H-2D(d) receptor) or anti-H-2D(d), mAb. Flow cytometric analysis revealed specific binding of H-2K(d) molecules to AIM (K(d)=2.7x10(-9) M); and the binding was completely suppressed by the addition of R12 mAb. These results demonstrate that a novel receptor (MMR2) for H-2K(d) molecules was induced on effector macrophages responsible for allograft (H-2D(d)K(d)) rejection by H-2D(b)K(b) mice.

Regulation of hair regrowth in alopecic site of IFN-gamma-/- mice by macrophages infiltrating into allograft in IFN-gamma+/+ mice.

We previously demonstrated that around 6 weeks of age, most of the interferon-gamma (IFN-gamma)-/- C57BL/6 mice began to lose morphogenesis-derived hairs in their dorsal and occipital areas and that hair regrowth in the alopecic site was induced by intraperitoneal (i.p.) injection of IFN-gamma and allogeneic Meth A cells. Here, we explored the IFN-gamma mRNA expression in the cells infiltrating into allograft in IFN-gamma(+)/(+) mice by RT-PCR and adoptively transferred specific antigen-minus infiltrates into IFN-gamma-/- mice to assess the hair regrowth inducibility. IFN- gamma mRNA was expressed in the infiltrates on days 3-8 after allografting, with a peak on day 3 or 4, and CD4(+) and F4/80(+) cells were the major producers of IFN-gamma. All infiltrates on day 3 induced hair regrowth, whereas those on days 0-2 or 4-8 were ineffective or partially effective, respectively. The removal of F4/80(+) macrophages from all infiltrates failed to induce hair regrowth, whereas the removal of Ly-6C(+) macrophages rather accelerated the hair regrowth. These results showed that F4/80(+), Ly-6C(+), and CD4(+) and F4/80(+) cells were stimulatory, inhibitory, and IFN-gamma-producing cells, respectively, in the regulation of hair regrowth.

IFN-gamma: a cytokine essential for rejection of CTL-resistant, virus-infected cells.

We recently demonstrated differential susceptibility of cells expressing viral antigen to killing by antigen-specific cytotoxic T lymphocytes (CTLs). In addition, interferon-gamma (IFN-gamma) has been implicated in the clearance of some viruses from tissues. We explored the role of IFN-gamma in the cytotoxicity of Sendai virus-specific CTLs against virus-infected RL(male symbol)1 (T cell leukemia) or Meth A (fibrosarcoma) cells, as well as the growth of subcutaneously (s.c.) transplanted, virus-infected cells in IFN-gamma(+/+) or IFN-gamma(/) mice of the syngeneic strain (BALB/c). Sendai virus-specific CTLs were cytotoxic against virus-infected RL(male symbol)1 cells, and s.c. transplanted, virus-infected RL(male symbol)1 cells were acutely rejected from IFN-gamma(+/+) or IFN-gamma(/) mice. In contrast, the CTLs were inactive toward virus-infected Meth A cells, but s.c. transplanted, virus-infected Meth A cells were acutely rejected from IFN-gamma(+/+) but not IFN-gamma(/) mice. The s.c. growth of virus-infected Meth A cells in the mutant mice was markedly inhibited by s.c. injections of IFN-gamma, and the rejection from IFN-gamma(+/+) mice was delayed after specific elimination of macrophages by intravenous (i.v.) injections of dichloromethylene diphosphonatecontaining liposomes. These results suggest an essential role of IFN-gamma and involvement of macrophage in the rejection of CTL-resistant, virus-infected cells.

Endolymphatic perfusion with EGTA-acetoxymethyl ester inhibits asphyxia- and furosemide-induced decrease in endocochlear potential in guinea pigs.

We examined the effect of the Ca(2+) concentration in the endolymph ([Ca](e)) or in the endolymphatic surface cells ([Ca](i)) on the endocochlear potential (EP) by using an endolymphatic or perilymphatic perfusion technique, respectively. (i) A large increase in [Ca](e) up to approximately 10(-3) M with a fall in the EP was induced by transient asphyxia ( approximately 2 min) or by the intravenous administration of furosemide (60 mg/kg), and a significant correlation was obtained between the EP and p[Ca](e) (= -log [Ca](e), r = 0.998). (ii) Perfusion of the endolymph with 10 mM EGTA for 5 min neither produced any significant change in the EP nor altered the asphyxia-induced change in EP (DeltaEP(asp)), suggesting that neither [Ca](e) nor the Ca(2+) concentration gradient across the stria vascularis contributed directly to the generation of the EP in the condition of low [Ca](e). In contrast, endolymphatic perfusion with high Ca(2+) (more than 10 mM) produced a decrease in EP and a significant correlation was obtained between the EP and the Ca(2+) concentration of perfusion solution (r = 0.982), suggesting that Ca(2+) permeability may exist across the stria vascularis. (iii) The administration of a Ca(2+) chelator, EGTA-acetoxymethyl ester (AM, 0.3 mM), to the endolymph, which produced a gradual increase in EP, suppressed significantly, by 60-80%, DeltaEP(asp) or furosemide-induced changes in EP. In contrast, perilymphatic administration of 0.5 mM EGTA-AM caused no significant suppression of the DeltaEP(asp). These findings suggest that [Ca](i) plays an important role in generating/maintaining a large positive EP.

Blood supply--susceptible formation of melanin pigment in hair bulb melanocytes of mice.

BACKGROUND: Allogeneic skin grafts onto C57BL/6 mice are rejected, and the rejected skin is replaced by surrounding skin with black hair. In contrast, syngeneic skin grafts are tolerated, and gray hair grows on the grafts. METHODS: To explore the mechanism of gray hair growing on the tolerated skin grafts, we prepared full-thickness skin (2-cm square) autografts, 2 (2 cm + 2 cm) horizontal or vertical parallel incisions, and U-shaped (2 cm × 2 cm × 2 cm) flaps with or without pedicle vessels. The grafts, incisions, and flaps were fixed by suturing with string and protected by a transparent bandage. On day 14 after the operation, the bandages were removed to observe the color of the hair growing on the skin. RESULTS: Skin autografts from wild-type or hepatocyte growth factor-transgenic (Tg) C57BL/6 mice survived with gray hair, whereas those from steel factor (Kitl)-Tg C57BL/6 mice survived with black hair. In addition, U-shaped flaps lacking both of the 2 main feeding vessels of wild-type mice had gray hair at the tip of the flaps. Light microscopy after staining with hematoxylin and eosin or dihydroxyphenylalanine showed that the formation of melanin pigment in the follicles, but not in the interadnexal skin, was susceptible to the blood supply. CONCLUSIONS: Melanin pigment formation in the hair bulb melanocytes appeared to be susceptible to the blood supply, and melanocytosis was promoted in the follicles and in the epidermis of Kitl-Tg C57BL/6 mice.

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-2D(b)K(b)) macrophage-mediated, cell-to-cell contact-dependent, d haplotype-specific lysis of allografts (e.g., BALB/c skin and Meth A cells; H-2D(d)K(d)) in the rejection site and isolated two cDNA clones encoding receptors on macrophages for H-2D(d) and H-2K(d), 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 D(d), K(d), or D(d)K(d) 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 K(d) transgenic skin grafts. Surprisingly, they also lacked MMR1 mRNA and protein expression in their monocytes and failed to reject D(d) or D(d)K(d) transgenic skin grafts. However, they did reject skin grafts from mice expressing H-2I(d), minor H(d), or third-party major histocompatibility complex. On the contrary, D(d)-, K(d)-, or D(d)K(d)-EL-4 cells injected intradermally or intraperitoneally into MMR2 KO mice were rejected by TCR(αß)(+)/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.

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.

IL-4-dependent induction of IgE basophils in peripheral blood and IgE B cells in spleen as respective indicators of allergen sensitization and a precursor of cells secreting allergen-specific IgE antibody.

It was recently reported by us that either primary i.n. or i.p. injection of cedar pollen extract into BALB/c mice, or a second s.c. injection of the allergen into i.v. or s.c. sensitized mice, causes an IL-4-dependent increase in total IgE serum antibody to produce allergen-specific IgE antibody upon further s.c. sensitization. To determine the biology of total IgE antibody, in the present study IgE+ cells in peripheral blood or lymphoid tissues of allergen-sensitized BALB/c mice have been characterized. In peripheral blood, mice sensitized one to three times with the allergen produced a 2.5- to 4-fold increase in the number of IgE+ cells, with a time-course similar to that of the concentration of total IgE antibody in serum. These IgE+ cells were basophils. On the other hand, the number of IgE+ cells in the lymphoid tissues did not change significantly after an i.n., i.p., i.v. or s.c. injection of allergen into the mice, whereas a second s.c. injection of the allergen into the i.v.-, but not into the i.n.-, i.p.- or s.c.-, sensitized mice induced a small number of IgE+/IgM+/B220+ B cells in the spleen. In contrast, IgE+ cells were not seen in the blood or spleen of IL-4 -/- mice after sensitization with the allergen. These results suggest that IgE+ basophils in the peripheral blood, and IgE+ B cells in the spleen, might be IL-4-dependently induced as an indicator of sensitization with allergen, and a precursor of cells secreting allergen-specific IgE antibody, respectively.

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 beta(2)M, 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-2(d)) skin onto or Meth A (H-2(d)) tumor cells into C57BL/6 (H-2(b)) 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-2D(d)K(d) 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).

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-alpha 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.

Physiological role of L-type Ca2+ channels in marginal cells in the stria vascularis of guinea pigs.

Using immunohistochemical and electrophysiological methods, we investigated the role of L-type Ca(2+) channels in the regulation of the endocochlear potential (EP) of the endolymphatic surface cells (ESC) of the guinea pig stria vascularis. The following findings were made: (1) Administration of 30 microg/ml nifedipine via a vertebral artery significantly suppressed the transient asphyxia-induced decrease in the EP (TAID) and the transient asphyxia-induced increase in the Ca(2+), referred to as TAIICa, concentration in the endolymph ([Ca](e)). (2) The endolymphatic administration of 1 microg/ml nifedipine significantly inhibited the TAID as well as the TAIICa. The endolymphatic administration of nifedipine (0.001-10 microg/ml) inhibited the TAID in a dose-dependent manner. (3) The endolymphatic administration of (+)-Bay K8644, an L-type Ca(2+) channel closer, significantly inhibited the TAID, whereas (-)-Bay K8644, an L-type Ca(2+) channel opener, caused a large decrease in the EP from approximately +75 mV to approximately +20 mV at 10 min after the endolymphatic administration. (4) By means of immunohistochemistry, a positive staining reaction with L-type Ca(2+) channels was detected in the marginal cells of the stria vascularis. (5) Under the high [Ca](e) condition, we examined the mechanism of the TAIICa and hypothesized that the TAIICa might have been caused by the decrease in the EP through a shunt pathway in the ESC. (6) The administration of nifedipine to the endolymph significantly inhibited the Ba(2+)-induced decrease in the EP. These findings support the view that L-type Ca(2+) channels in the marginal cells regulate the EP, but not directly the TAIICa.

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 (approximately 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-2(d))-type lymphoblasts, suggesting H-2(d)-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-2(d)-specific effector cells for allografted Meth A cells and are a more fully activated macrophage with unique morphological features.