Role of osteopontin, a multifunctional protein, in allergy and asthma.

Osteopontin (OPN) is an extracellular matrix protein and immune modulator with a wide range of functions. OPN is recognized as a key cytokine in Th1 immune responses, yet its potential involvement in allergic/asthmatic responses has been investigated only recently. Current data from molecular and cellular studies and studies of OPN-deficient mice provide evidence that OPN plays multiple roles in the regulation of allergic responses, including regulation of IgE response, inflammatory cell migration, and the development of airway fibrosis and angiogenesis. These results suggest that OPN is a pleiotropic cytokine that functions both systemically and locally in tissue mucosa. Notably, OPN is able to exert its effects through different functional domains, and the secreted and intracellular forms of OPN may have distinct functions. Future research to elucidate all aspects of OPN function is needed to ultimately establish its role in the regulation of immune responses and various disease processes, including those critically involved in the development of allergies and asthma.

A monoclonal antibody (8H3) that binds to rat T lineage cells and augments in vitro proliferative responses.

A murine monoclonal antibody, designated 8H3, recognizes a cell surface antigen expressed exclusively on rat T lineage cells. 8H3 antibody immunoprecipitated 180-, 120-, and 90-kD components from rat thymocytes as well as splenic T cells under nonreducing conditions. 8H3 antibody specifically inhibited the binding of thymocytes to fibronectin. Furthermore, binding of rat thymocytes to immobilized synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro-Cys-BSA was inhibited by 8H3 antibody as was Gly-Arg-Gly-Asp-Ser-Pro-Cys, but not by Gly-Arg-Ala-Asp-Ser-Pro-Lys or Gly-Arg-Gly-Glu-Ser-Pro. Crosslinking of 8H3 antigen on double-negative thymocytes and adult thymocytes, as well as splenic T lymphocytes by 8H3 antibody and F(ab')2 fragments of goat antibodies to mouse immunoglobulin, led to an increase in the concentration of cytoplasmic free Ca2+ due to the release of Ca2+ from intracellular stores as well as the influx of Ca2+ from extracellular sources. Expression of interleukin 2 receptor and subsequently cell proliferation was observed upon incubation of thymocytes and splenic T cells with 8H3 antibody. Furthermore, 8H3 antibody induced the proliferation of double-negative thymocytes. These data collectively indicated that a cell surface antigen, 8H3, is involved in not only cell adhesion but also involved in the expression of immature as well as mature thymocytes.

Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4.

This report shows that cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) plays a key role in T cell-mediated dominant immunologic self-tolerance. In vivo blockade of CTLA-4 for a limited period in normal mice leads to spontaneous development of chronic organ-specific autoimmune diseases, which are immunopathologically similar to human counterparts. In normal naive mice, CTLA-4 is constitutively expressed on CD25(+)CD4(+) T cells, which constitute 5-10% of peripheral CD4(+) T cells. When the CD25(+)CD4(+) T cells are stimulated via the T cell receptor in vitro, they potently suppress antigen-specific and polyclonal activation and proliferation of other T cells, including CTLA-4-deficient T cells, and blockade of CTLA-4 abrogates the suppression. CD28-deficient CD25(+)CD4(+) T cells can also suppress normal T cells, indicating that CD28 is dispensable for activation of the regulatory T cells. Thus, the CD25(+)CD4(+) regulatory T cell population engaged in dominant self-tolerance may require CTLA-4 but not CD28 as a costimulatory molecule for its functional activation. Furthermore, interference with this role of CTLA-4 suffices to elicit autoimmune disease in otherwise normal animals, presumably through affecting CD25(+)CD4(+) T cell-mediated control of self-reactive T cells. This unique function of CTLA-4 could be exploited to potentiate T cell-mediated immunoregulation, and thereby to induce immunologic tolerance or to control autoimmunity.

Osteopontin is involved in the initiation of cutaneous contact hypersensitivity by inducing Langerhans and dendritic cell migration to lymph nodes.

Osteopontin (OPN) is a chemotactic protein that attracts immune cells, to inflammatory sites. The sensitization phase of allergic cutaneous contact hypersensitivity (CHS) requires the migration of Langerhans cells/dendritic cells (LCs/DCs) from skin to draining lymph nodes. Characterizing OPN function for LC/DC migration we found upregulated OPN expression in hapten sensitized skin and draining lymph nodes. OPN induces chemotactic LC/DC migration, initiates their emigration from the epidermis, and attracts LCs/DCs to draining lymph nodes by interacting with CD44 and alphav integrin. Furthermore, OPN-deficient mice have a significantly reduced CHS response that correlates with an impaired ability of OPN-deficient mice to attract LCs/DCs to draining lymph nodes. In conclusion, OPN is an important factor in the initiation of CHS by guiding LCs/DCs from skin into lymphatic organs.

Osteopontin is involved in migration of eosinophils in asthma.

BACKGROUND: Osteopontin (OPN) is an extracellular matrix protein with a wide range of functions, and is involved in various inflammatory diseases. However, the role of OPN in eosinophilic airway inflammation is unclear. OBJECTIVE: To elucidate the role of OPN in eosinophilic airway inflammation. METHODS: OPN protein levels in induced sputum from asthmatic patients and healthy controls were measured. Eosinophil migration assays were performed in the presence or absence of OPN, a blocking antibody (Ab) recognizing its integrin-binding domain (2K1) and an anti-integrin alpha 4 Ab (P1H4). In the mouse asthma model, the levels of eosinophilia were examined in bronchoalveolar lavage fluids (BALFs) from ovalbumin (OVA)-sensitized and -challenged mice with or without administration of an Ab (M5) corresponding to human 2K1. RESULTS: Levels of OPN in induced sputum were significantly higher in asthmatic patients when compared with healthy controls. In addition, levels of OPN were correlated with the percentage of sputum eosinophils. OPN induced significant migration of human eosinophils and this effect was inhibited by 2K1 and P1H4. M5 significantly attenuated OVA-induced eosinophilia in BALFs. CONCLUSION: These results indicate that OPN plays a role in the migration of eosinophils into the airways and may be involved in the pathogenesis of asthma.

Peptide mimics of the CTLA4-binding domain stimulate T-cell proliferation.

Phage library clones selected by a conformational epitope-recognizing and inhibitory monoclonal antibody may display moieties that mimic a receptor/ligand-like three-dimensional structure. This pseudoreceptor/ligand should be able to bind to natural ligand/receptor molecules. We tested this idea using anti-T cell costimulatory molecule antibodies and successfully isolated phage clones with costimulatory effects on T-cell proliferation. This strategy facilitates the designing of regulatory peptide molecules in the absence of precise information about the structure-function relationships in receptor/ligand interactions.

Monoclonal antibodies that detect different antigenic determinants of the same human osteosarcoma-associated antigen.

Monoclonal antibodies (MoAbs) against human osteosarcoma cells were obtained by the fusion of NS/1 mouse myeloma cells with spleen cells from the human osteosarcoma cell line-immunized BALB/c mice. Two hybrid clones were established and designated as 2H10 and 2D3. Both MoAbs reacted strongly with all osteosarcoma tissues but not with other bone and soft tissue tumors such as chondrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, and rhabdomyosarcoma. In addition, neither MoAb reacted with tumor cell lines and tissues obtained from other cancers. Immunohistochemical analysis demonstrated that 2H10 and 2D3 reacted with endothelial cells in sarcoma tissues, but not with those of other tumors and normal tissues. 2H10 also reacted with cells on the basal layer of epidermis of the skin. 2H10- and 2D3-defined antigen has an approximate molecular weight of 75,000 under nonreducing and reducing conditions, indicating that the antigen has a single chain structure and there is no intramolecular disulfide bond. 2H10- and 2D3-defined antigen has a pI value between 5.5 and 6.2. Sequential immunoprecipitation analysis clearly demonstrated that 2H10 and 2D3 recognized the same antigen molecule. However, further analysis suggested the possibility that 2H10 and 2D3 MoAbs recognized the different antigenic determinants on the same antigen molecule.

New monoclonal antibody, 1C5, reactive with human cervical adenocarcinoma of the uterus, with immunodiagnostic potential.

A murine monoclonal antibody, 1C5, was produced by fusion of spleen cells obtained from mice immunized with CAC-1, a human cell line of adenocarcinoma derived from uterine cervix, and NS/1 myeloma cells. 1C5 can be used for the staining of routine formalin-fixed and paraffin-embedded tissue sections. 1C5-defined antigen was found to have a molecular weight of 26,000. The 1C5-defined antigen was resistant to neuraminidase and trypsin treatment, but sensitive to periodate treatment, indicating that an epitope of the 1C5-defined antigen is a carbohydrate moiety. Immunohistochemical study using immunoperoxidase staining demonstrated that 1C5 reacted with 87% of adenocarcinomas of the uterine cervix, 39% of endometrial carcinomas of the uterus, 100% of ovarian mucinous cystadenocarcinomas, 43% of ovarian serous cystadenocarcinomas, 45% of adenocarcinomas of the colon, and 40% of gastric adenocarcinomas, thus showing the broad reactivity to adenocarcinoma cells of various origins. However, 1C5 did not show any reactivity to ectocervix epithelium, cervical intraepithelial neoplasia, or squamous cell carcinoma of the uterine cervix. In addition, adenocarcinoma of the uterine cervix exhibited strong cytoplasmic reactivity with 1C5, whereas endometrial carcinoma of the uterus showed the luminal reactivity. 1C5 also reacts with 95% ethanol-fixed malignant cells in cervical smears.

Lymphoid cell subpopulations infiltrating into autologous rat tumors undergoing rejection.

Lymphoid cell subpopulations infiltrating into autografts of methylcholanthrene-induced sarcomas in rats immunized with autologous tumor cells were identified in terms of immunohistochemical and cytofluorographic techniques using various monoclonal antibodies raised against different classes of rat lymphohemopoietic cells. These antibodies included in this study directed to rat T-cell antigens corresponding to mouse Lyt-1 (RLyt-1) and Lyt-2,3 antigens (RLyt-2) and to W3/25 antigen expressed on a particular subset of rat T-cells with helper function, as well as to rat granulocyte-macrophage-specific antigen (RGM-1). Histological studies demonstrated that the autografts of highly antigenic tumors introduced to the primary hosts were completely rejected following massive immigration of lymphoid cells into the tumor sites, which was not observed in progressively growing, minimally antigenic tumors. These lymphoid cells found within regressing highly antigenic tumor autografts were identified mostly to be T-cells bearing RLyt-1 (approximately 70%), and more than two-thirds of these T-cells expressed RLyt-2 antigen. In contrast to T-cells, macrophages and B-cells, each of which could be recognized by the presence of either RGM-1 antigen or immunoglobulin on their cell surfaces, appeared to have a minimal role in the rejection of autochthonous tumors, as reflected by their less frequent appearance within the tumor tissues during the rejection process.

CD44 variants but not CD44s cooperate with beta1-containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis.

The expression of osteopontin (OPN), CD44 variants, and integrins has been correlated with tumorigenesis and metastasis. Here we show that these proteins cooperate to enhance cell motility. First, we demonstrate that several different CD44 variants bind to OPN in an arginine-glycineaspartic acid-independent manner, but that the standard form of CD44 does not. These CD44 variants bind to both the amino- and COOH-terminal portions of OPN independently of the arginine-glycine-aspartic acid sequence, suggesting that multiple domains on OPN can be bound by the CD44 variants. Antibodies directed against the integrin beta1 subunit are able to inhibit this binding. The binding of CD44 variants to OPN is significantly augmented by both anti-CD44s and anti-CD44v antibodies. This augmentation by anti-CD44 antibodies is OPN specific and, again, can be blocked by anti-beta1 antibodies. Finally, we show that OPN binding by CD44 variants/beta1-containing integrins promotes cell spreading, motility, and chemotactic behavior.

High plasma concentrations of osteopontin in patients with interstitial pneumonia.

Osteopontin (OPN) produced by alveolar macrophages functions as a fibrogenic cytokine in the development of bleomycin (BLM)-induced murine pulmonary fibrosis, and OPN mRNA is expressed on lung tissues from patients with idiopathic pulmonary fibrosis (IPF). The present study investigates plasma OPN levels in human interstitial pneumonia (IP) and their relationships with disease severity by analyzing the correlation between plasma OPN concentrations and pulmonary functions. The concentrations of OPN in plasma were measured in 17 patients with IP, in 9 with sarcoidosis and in 20 healthy controls using an antigen-capture enzyme-linked immunosorbent assay. The concentrations of OPN in plasma were significantly higher in IP patients than in those with sarcoidosis or in controls. Based on a Receiver Operating Characteristic curve analysis, cut-off points between 300 and 380 ng/ml discriminated between IP and control subjects with 100% sensitivity and 100% specificity. In such case, the sensitivity for sarcoidosis decreased (55.5-33.3%) in cut-offs with 100% specificity. Plasma OPN levels inversely and closely correlated with arterial oxygen tension (PaO2) in patients with IP. Immunohistochemically, OPN was localized predominantly in macrophages and airway epithelium. These findings suggest that plasma OPN levels were found to be associated with the presence of IP, and that OPN play an important role in the development of IP.

Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG.

Adenovirus vectors can transfer recombinant genes efficiently into a wide variety of cells in vivo, but have serious limitations: gene expression is transient and secondary gene transfer is inefficient or impossible because of cellular and humoral immune responses against adenovirus-transduced cells. To solve these limitations, we have constructed an adenovirus vector, Adex1CACTLA4IgG, that expresses CTLA4IgG molecules. After in vivo administration of Adex1CACTLA4IgG (9.0 x 10(9) PFU), the peak level of serum CTLA4IgG was 29.8 mg/ml on day 4. The serum CTLA4IgG concentration gradually fell but was still 5.7 mg/ml on day 90. However, the serum concentration of CTLA4IgG was elevated after a second administration of Adex1CACTLA4IgG. The production of antibody against adenovirus was completely prevented after treatment with Adex1CACTLA4IgG. In addition, coadministration of Adex1CALacZ with Adex1CACTLA4IgG induced persistent hepatic expression of beta-Gal molecules, while administration of Adex1CALacZ alone induced transient expression of beta-Gal molecules. More importantly, on day 160 a secondary challenge with Adex1CALacZ was possible in mice treated with Adex1CALacZ plus Adex1CACTLA4IgG. Thus, we have demonstrated that (1) gene expression of a recombinant adenovirus, Adex1CACTLA4IgG, is persistent in liver and secondary administration of this adenovirus is possible, (2) coadministration of Adex1CACTLA4IgG virus with another adenovirus, AdexCALacZ, prolongs AdexCALacZ-mediated gene expression, and (3) Adex1CACTLA4IgG is useful for secondary challenge with Adex1CALacZ.

Long-term acceptance of allografts by in vivo gene transfer of regulatable adenovirus vector containing CTLA4IgG and loxP.

CTLA4IgG was shown to inhibit the costimulatory signal for T cell activation by interfering with the ligation of CD28 and B7-1 or B7-2. To inhibit various immune responses including acute cellular rejection of allografts, a certain level of serum CTLA4IgG should be maintained for an appropriate period. We previously reported on an adenovirus vector containing CTLA4IgG, which we designated Adex1CACTLA4IgG. Adex1CACTLA4IgG was able to maintain a significant level of serum CTLA4IgG for a long period on intravenous injection, which in turn inhibited various immune responses including protective immunity against infectious agents. To overcome the inhibitory effect, we constructed a new adenovirus vector, Adex1CALoxCTLA4IgGLox, by cloning CTLA4IgG cDNA between two loxP sequences under the control of the CAG promoter. We demonstrated that the administration of adenovirus vector containing Cre recombinase gene (Adex1CACre) at the desired time induced Cre-mediated recombination within a gene derived from Adex1CALoxCTLA4IgGLox vector, and the cDNA of CTLA4IgG was excised from the transduced gene and terminated the expression of CTLA4IgG in vitro and in vivo. More importantly, we also demonstrated that the long-term acceptance of allografts was achieved after the termination of CTLA4IgG expression, while the immune response against adenovirus was restored.

Successful gene therapy via intraarticular injection of adenovirus vector containing CTLA4IgG in a murine model of type II collagen-induced arthritis.

We previously constructed an adenovirus vector carrying a gene encoding a soluble form of fusion protein, consisting of the extracellular portion of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglobulin G1 (Adex1CACTLA4IgG). Murine type II collagen-induced arthritis (CIA) was treated with Adex1CACTLA4IgG. A single intraarticular injection of 1 x 10(5) PFU was able to support serum CTLA4IgG at more than 10 microg/ml for at least 12 weeks and was able to inhibit the CIA clinically and histologically. In contrast, intravenous, intramuscular, or subcutaneous injection of 1 x 10(5) PFU was unable to support a significant level of serum CTLA4IgG and thus was unable to inhibit the development of arthritis. Thus, we demonstrated that (1) a low-dose intraarticular injection of Adex1CACTLA4IgG was effective in delaying the onset of CIA and reducing the severity of arthritis; (2) an intraarticular (knee joint) injection of Adex1CACTLA4IgG effectively blocked the development of arthritis in distal paws; (3) the inhibitory effect of Adex1CACTLA4IgG lasted at least up to 20 weeks; (4) although serum CTLA4IgG at more than 10 microg/ml persisted for at least 12 weeks, mice treated by intraarticular injection of Adex1CACTLA4IgG were not anergic to adenovirus and were able to mount antibody responses against various antigens.

Vaccination with B7-1+ tumor and anti-adhesion therapy with RGD pseudo-peptide (FC-336) efficiently induce anti-metastatic effect.

We have previously shown that expression of costimulatory ligand B7-1 on MHC class I+ tumor cells (B16-BL6 melanoma) resulted in marked reduction of lung metastasis caused by i.v. injection into immunocompetent syngeneic mice and led to induction of immunity to the challenge by the parental B7-1 negative tumor. Here we investigated the effectiveness of irradiated B7-1 transfected tumor cells as a vaccine on established tumor metastasis and whether or not expression of B7-1 molecule on tumor cells in combination with administration of anti-adhesion peptide FC-336 can augment the antimetastatic efficacy. Immunization with X-irradiated B7-1 transfectants after i.v. injection of B7-1- parental B16-BL6 cells was effective in inhibiting lung metastasis. We also found that vaccination with irradiated B7-1 transfectants after excision of primary tumor on day 21 resulted in significant inhibition of spontaneous lung metastasis by intrafootpad injection of viable parental B16-BL6 melanoma, as compared with the untreated control. However, immunizing twice with mock transfectants did not affect inhibition of spontaneous lung metastasis of wild-type tumors. On the other hand, multiple administration of a pseudo-peptide of RGD sequence (FC-336) after tumor inoculation inhibited spontaneous lung metastasis through the interference of tumor invasion, migration and adhesion. Combined treatment of B7-1 transfected tumor vaccine and anti-adhesive therapy with FC-336 led to the augmentation of the antimetastatic effect in both experimental and spontaneous metastasis models, as compared with either treatment alone. B7-1- and FC-336-mediated inhibition of tumor metastasis may be mediated by different mechanisms at various steps of metastasis, based on the regulation (promotion or inhibition) of tumor interaction with host cells and components.

CTLA4IgG treatment induces long-term acceptance of rat small bowel allografts.

BACKGROUND: CTLA4 immunoglobulin (Ig)G that binds to B7 effectively inhibits the signaling of CD28/CTLA4-B7 pathway and induces antigen specific T cell unresponsiveness in vitro and in vivo. Using CTLA4IgG, we examined induction of long-term graft survival and the mechanism of maintenance of tolerance in rat allogeneic small bowel transplantation. METHODS: Small bowels of Brown-Norway rats (RT1n) were heterotopically transplanted into Lewis rats (RT1l). Recipients were treated with an i.p. injection of either CTLA4IgG or control IgG for 7 days. RESULTS: Long-term survival was observed in rats treated with CTLA4IgG, whereas control rats died within 16 days after transplantation. To examine whether a tolerant state was established in long-term survival rats, secondary transplantation was performed using small bowels of Brown-Norway rats or ACI (RT1b) rats. It was demonstrated that small bowels of Brown-Norway rats were accepted; however, those of ACI rats were rejected within 10 days. Serum concentrations of interleukin (IL)-4 were maintained at >50 microg/ml for 7 days after transplantation in rats treated with CTLA4IgG but <15 microg/ml in control rats. IL-2 concentration was reduced to half in CTLA4IgG-treated rats compared with that in control recipients. Serum IFN-gamma in CTLA4IgG-treated recipients increased after transplantation and was not distinguishable from that of control recipients during the first 7 days after transplantation. Conclusion. We demonstrated that CTLA4IgG treatment alone for 7 days induced a long-term donor specific tolerance in rat allogeneic small bowel transplantation. The induction of long-term acceptance of small bowel allografts by CTLA4IgG is not caused by simply the shift of anti-alloimmune responses from Thl to Th2 cytokine production.

Blocking the CD28-B7 T-cell costimulatory pathway abrogates the development of obliterative bronchiolitis in a murine heterotopic airway model.

BACKGROUND: CTLA4IgG that binds to B7 effectively inhibits the signaling of CD28/B7 pathway and induces antigen-specific T-cell unresponsiveness in vitro and in vivo. We examined whether the development of obliterative bronchiolitis in a murine heterotopic airway transplantation model is T cell dependent and whether CTLA4IgG abrogates the development of obliterative bronchiolitis. METHODS: Tracheae with main bronchi from C3H/He (H2k), BALB/C (H2d), or C57BL/6 (H2b) mice were transplanted heterotopically into subcutaneous pockets on the backs of BALB/C or BALB/C nu/nu mice on day 0. Recipient mice were untreated or intraperitoneally treated with either CTLA4IgG or human IgG with different time and dose schedules. RESULTS: The development of obliterative bronchiolitis, which leads to luminal obliteration by fibrous tissue in a murine heterotopic airway transplantation model, was T cell dependent and the development of obliterative bronchiolitis was significantly abrogated by the CTLA4IgG treatment. However, the normal ciliated columnar respiratory epithelial cells in allografts were lost and replaced by flattened attenuated epithelial cells even after the CTLA4IgG treatment. We further demonstrated that CTLA4IgG treatment did not result in the induction of donor-specific unresponsiveness. CONCLUSIONS: We demonstrated that the development of obliterative bronchiolitis in a murine heterotopic airway model involves both CD28/B7-dependent and -independent processes. The luminal obliteration by fibrous tissue is clearly CD28/B7 dependent and can be inhibited by CTLA4IgG. The luminal obliteration of allografted trachea by fibrous tissues and the loss of ciliated columnar respiratory epithelial cells represent distinct disease processes.

Demonstration of the interaction between the CD23 molecule and the galactose residue of glycoproteins.

The CD23 molecule is a low-affinity receptor for IgE and has a marked homology in amino acid sequence with C-type animal lectins, including asialoglycoprotein receptor. We tested whether the CD23 antigen can indeed interact with the sugar chain of glycoproteins. Detergent extract of the membrane component from Epstein-Barr Virus (EBV)-transformed human B-cell line, L-KT9 cells, was incubated with asialofetuin (ASF)-coupled Sepharose, and bound proteins were effectively eluted by 0.3 M lactose or galactose which were among the competitive sugars tested. In this eluate, the CD23 molecule was detected by an immunoblotting technique. Because fetuin has both an N- and O-type sugar chain on the molecule, we tested which type of sugar chain can interact with CD23. The CD23 molecule interacted with asialocasein having a sugar chain with the Gal-GalNAc structure with asialobovine submaxillary mucin having the GalNAc structure, and also with ASF; however, it faintly interacted with ASF after removal of the O-type sugar chain by beta-elimination. These results showed that the CD23 molecule can, indeed, interact with the galactose residue, especially with the Gal-GalNAc rather than the Gal-GlcNAc structure of the terminal sugar chain of glycoproteins.

The rat neutrophil low-affinity Fc receptor for IgG: molecular cloning and functional characterization.

A complementary DNA (cDNA) clone encoding rat Fc gamma receptor II (Fc gamma RII) was isolated from rat neutrophils and characterized. The cDNA encodes a type I transmembrane protein with 285 amino acids having an extracellular domain consisting of two immunoglobulin-like domains (179 amino acids), a transmembrane domain (26 amino acids), and a cytoplasmic domain (47 amino acids). The nucleotide sequences are identical to that of recently cloned Fc gamma RII from rat mast cells. This protein was expressed on FcR-negative Chinese hamster ovary (CHO) cells. The characterization of cDNA-transfected CHO cells clearly indicated that the protein encoded by the cDNA clone binds guinea-pig IgG1 and IgG2 complexes and unexpectedly binds monomeric rat IgG1, but not IgG2. Furthermore, the affinity for immune complexes was significantly augmented by protease treatment of transfectants. In addition, endocytosis of immune complex was noted in transfectants.

The dysfunction of calcium-ATPase pump in double negative T cells of autoimmune-prone mice.

Double negative (DN) T cells expanding in peripheral lymphoid tissues in mice bearing lymphoproliferation (lpr) gene are generally unresponsive to mitogens, antigens, and anti-T cell receptor (TCR) or anti-CD3 monoclonal antibodies (mAb). In response to the stimulation with 0.125-5.0 microM ionomycin, control T cells sustained an increase in intracellular free calcium ([Ca2+]i), while DN lpr T cells showed a gradual fall following initial rapid increase in [Ca2+]i. Such gradual fall in [Ca2+]i was overcome by the addition of endoplasmic and sarcoplasmic reticulum Ca(2+)-ATPase inhibitor or high dose (10 microM) of ionomycin. The requirement of high concentration of calcium ionophore for the sustained increase of [Ca2+]i in lpr DN T cells is due to dysfunction of Ca(2+)-ATPase pump.