The cryopreservation of immunocompetent cells

Lymphoid cells sensitized against tumor cells in vivo were removed from sheep and mice, frozen and thawed, and then assayed for retention of cytotoxic function. Sheep lymphocytes efferent from immunized nodes have been shown previously to be cytotoxic owing to the production of antibody and these cells retained approximately 50% of their activity after freezing. The specific cytotoxicity of sensitized mouse spleen cells has been shown to be due to thymus-derived lymphocytes and such cells totally retained specific cytotoxic action after freezing. However, spleen cells also generally exhibited a degree of nonspecific cytotoxic action which disappeared on freezing and was attributed to the selective elimination of glass-adherent cells by the freezing process.     

Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro

An in vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in vivo rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4 °C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement.Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and ⁸⁹Sr both are able to prevent rejection of marrow allografts in vitro. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with ⁸⁹Sr were effectively cytotoxic but lost practically all of their cytotoxicity afer incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in vivo during marrow allograft rejections.     

The in vitro killing of syngeneic cells by peritoneal cells from adjuvant-stimulated mice.

The cytotoxicity of peritoneal exudate cells from mice which had been injected with anaerobic coryneform organisms which have adjuvant activity was assessed by measuring the release of radioactive chromium from monolayers of whole mouse embryo cells. It was found that the peritoneal cells from adjuvant-stimulated mice were more cytotoxic than cells from normal mice. The increased cytotoxicity was present as early as 2 days after injection of the organisms, and was abolished by trypsin treatment of the peritoneal cells. The cytotoxic effect requires the presence of live peritoneal cells, and is more marked as the ratio of effector to target cells is increased. The plastic adherent cells of the peritoneal cell population are more effective in the cytotoxic reaction than are the non-adherent cells. The stimulated peritoneal cells can kill both syngeneic and allogeneic mouse embryo cells. Consideration is given to the possible mechanisms by which the increased cytotoxicity might be induced.     

Further studies on Th-B, a cell surface antigenic determinant present on mouse B cells, plasma cells and immature thymocytes.

A goat antiserum (Goat anti-M104E) has been produced which contains antibodies selectively cytotoxic for mouse B cells and a subpopulation of thymus cells. It reacts with the Th-B antigenic determinant which has been shown by us (1–3) to be present on B cells and on plasma cells and on some cells in the thymus. It also is very cytotoxic for mouse B cells while a previously developed rabbit antiserum was not. The antiserum was obtained by immunization with cells of the BALB/c mouse myeloma MOPC-104E. When the antiserum was purified by in vivo absorption in mice, antibodies remained which were cytotoxic for cells of all of several myelomas at a titer between 1:128 and 1:1024 as determined by an in vitro complement dependent cytotoxicity test. The in vivo purified antibodies were also cytotoxic for about 70% of thymus cells, for about 70% of spleen cells, for about 50% of lymph node cells and for about 20% of bone marrow cells. They were very cytotoxic for splenic or lymph node B cells separated from T cells by a nylon wool column and only slightly cytotoxic for splenic or lymph node T cells. The antibodies were only weakly cytotoxic for one out of five T cell tumors tested and not cytotoxic for the remaining four. Irrespective of target cells used, the cytotoxicity of purified Goat anti-M104E was easily removed by absorption with cell suspensions from tissues which contain B cells, plasma cells or thymus cells. In order to confirm that the same anti-Th-B antibodies recognize the determinant present on spleen cells and on some thymocytes, the purified Goat anti-M104E serum was absorbed with either spleen cells or thymus cells. The absorbed sera were tested for ability to label thymocytes or spleen cells using the fluorescence activated cell sorter (FACS). Either absorption removed essentially all the antibody capable of binding to either cell population. In addition it was shown, using the FACS, that only B cells and not T cells of the spleen contain the Th-B determinant. The anti-Th-B antibodies have now been used for the rapid elimination of B cells from a mixed population of lymphocytes without affecting the function of mature T cells. Thus in vitro treatment of spleen cells from SRBC-immunized donors with purified Goat anti-M104E plus complement results in the killing of a high proportion of the B memory cells as shown by the reduction of PFC produced when the treated cells are transferred to irradiated recipients. The T cell helper function of the transferred cells is not affected by Goat anti-M104E treatment as shown by appropriate cell transfer experiments in which effective B cells are provided by an AKR anti-Thy-1.2-treated spleen cell population and effective T cells are provided by the Goat anti-M104E-treated spleen cell population. Antibodies detecting Th-B may serve as an approach to understanding the ontogeny of lymphocytes. Our results suggest that Th-B is a cell surface marker appearing early in the development of lymphoid cells, on the common precursor of B and T cells and that it is lost from T cells as they mature in the thymus.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Histamine release from mouse and rat mast cells cultured with supernatants from chronic murine graft-vs-host splenocytes.

There is growing interest in studying pathways of mast cell activation. In a mouse model of chronic graft-vs-host disease (cGVHD) extensive mast cell activation and degranulation occurs in vivo coincident with the development of dermal fibrosis. An interesting feature of this model is that the mast cell reaction is slow to develop, occurring over a period of weeks and waning by 300 days. The aim of our work was to investigate the effects of supernatants from splenocytes of such cGVHD mice (cGVHD sups) on mouse and rat peritoneal mast cells cocultured with 3T3 skin fibroblasts. We found that cGVHD sups are able to release histamine from both mouse and rat cultured mast cells in a slow fashion. Histamine release became evident only after 5-8 days of coculture of the mast cells with the cGVHD supernatants and thereafter decreased to basal levels. Mast cell activation due to cGVHD supernatants was a noncytotoxic event as demonstrated by mast cell counts in the cocultures and by the ability of mast cells to exclude trypan blue. Mast cells that had been activated by incubation with the cGVHD sups were as responsive to stimulation with either anti-IgE antibodies or compound 48/80 as were mast cells incubated with control sups. Supernatants from mice early in GVHD (Days 11-28) were most active in promoting histamine release. Supernatants from spleens of mice which had GVHD for 290 days and where the mast cells had returned to full granulation in vivo were inactive. This is the first in vitro study demonstrating slow mast cell histamine release instituted by other cells, namely the splenocytes of cGVHD mice.     

Mast Cells and Gastrointestinal Dysmotility in the Cystic Fibrosis Mouse

Background

Cystic fibrosis (CF) has many effects on the gastrointestinal tract and a common problem in this disease is poor nutrition. In the CF mouse there is an innate immune response with a large influx of mast cells into the muscularis externa of the small intestine and gastrointestinal dysmotility. The aim of this study was to evaluate the potential role of mast cells in gastrointestinal dysmotility using the CF mouse (Cftr^tm1UNC, Cftr knockout).

Methodology

Wild type (WT) and CF mice were treated for 3 weeks with mast cell stabilizing drugs (ketotifen, cromolyn, doxantrazole) or were treated acutely with a mast cell activator (compound 48/80). Gastrointestinal transit was measured using gavage of a fluorescent tracer.

Results

In CF mice gastric emptying at 20 min post-gavage did not differ from WT, but was significantly less than in WT at 90 min post-gavage. Gastric emptying was significantly increased in WT mice by doxantrazole, but none of the mast cell stabilizers had any significant effect on gastric emptying in CF mice. Mast cell activation significantly enhanced gastric emptying in WT mice but not in CF mice. Small intestinal transit was significantly less in CF mice as compared to WT. Of the mast cell stabilizers, only doxantrazole significantly affected small intestinal transit in WT mice and none had any effect in CF mice. Mast cell activation resulted in a small but significant increase in small intestinal transit in CF mice but not WT mice.

Conclusions

The results indicate that mast cells are not involved in gastrointestinal dysmotility but their activation can stimulate small intestinal transit in cystic fibrosis.     

Bone marrow-derived inducible microglia-like cells ameliorate motor function and survival in a mouse model of amyotrophic lateral sclerosis

Background aimsAmyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease. Neuroinflammation in the spinal cord plays a pivotal role in the pathogenesis of ALS, and microglia are involved in neuroinflammation. Microglia mainly have two opposite phenotypes involving cytotoxic and neuroprotective properties, and neuroprotective microglia are expected to be a novel application for the treatment of ALS. Therefore, to establish a clinically applicable therapeutic method using neuroprotective microglia, the authors investigated the effect of inducing neuroprotective microglia-like cells from bone marrow for transplantation into ALS model mice.MethodsBone marrow-derived mononuclear cells were isolated from green fluorescent protein mice and cultured using different protocols of cytokine treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. Cells with a high potency of proliferation and differentiation into microglia were evaluated by gene analysis, flow cytometry and direct neuroprotective effects in vitro. These cells were named bone marrow-derived inducible microglia-like (BM-iMG) cells and transplanted into the spinal cords of ALS model mice, and behavioral tests, immunohistochemistry and gene expression profiling were performed.ResultsThree-day GM-CSF and 4-day GM-CSF + IL-4 stimulations were most effective in inducing BM-iMG cells from the bone marrow. Transplantation of BM-iMG cells improved motor function, prolonged survival and suppressed neuronal cell death, astrogliosis and microgliosis in the spinal cords of ALS mice. Moreover, neuroprotective genes such as Arg1 and Mrc1 were upregulated, whereas pro-inflammatory genes such as Nos2 and Il6 were downregulated.ConclusionsIntraspinal transplantation of BM-iMG cells demonstrated therapeutic effects in a mouse model of ALS. Further studies and clinical applications in patients with ALS are expected in the future.     

Suppressor factor produced by neonatal mouse spleen cells

Spleen cells from 5- to 6-day- old BDF₁ mice produce a soluble suppressor factor (SF) when cultured for 2–4 days in the presence of 10% fetal calf serum. This suppressor factor inhibits the mixed lymphocyte reactivity of adult mouse spleen cells as well as the in vitro generation of cytotoxic cells. The SF which is not H₂-restricted or antigen specific is most effective when added in the early phase of the culture period. The SF is resistant to heat and uv treatment and appears to consist of a large and small component. It is resistant to treatment with pronase or trypsin. The SF appears to be produced by neonatal spleen cells which are not adherent to plastic or Sephadex G-10 and are insensitive to treatment with anti-Thy 1.2 and complement. Incubation of SF with peritoneal exudate cells reduces suppressor activity.     

Cytotoxic activity of gypenosides and gynogenin against non-small cell lung carcinoma A549 cells

The activity of gypenosides and gynogenin of Gynostemma pentaphyllum against non-small cell lung carcinoma (NSCLC) A549 cells was investigated to identify the structural characteristics of gypenosides and gynogenin to have anti-NSCLC activity. Of the tested dammarane-type compounds, 20S-dammar-24-en-2α,3β,12β,20-tetrol showed the strongest activity against A549 cells. Based on the structure and cytotoxic activity relationships of gypenosides and gynogenin, the OH group in C-2, the connected sugar number and the configuration in C-20 were important for cytotoxic activity against A549 cells.     

Tumor necrosis factor-alpha dependent cytotoxicity of human skin mast cells is enhanced by anti-IgE antibodies

Mast cells dispersed from human skin and purified by density-gradient centrifugation were cytotoxic toward the mouse fibrosarcoma cell line WEHI-164. Skin mast cells were not cytotoxic toward the NK cell-sensitive cell line K562. Killing of WEHI-164 occurred over a prolonged (greater than 18 h) period of incubation with mast cells and was effectively inhibited by polyclonal antibodies and mAb against TNF-alpha suggesting that this cytokine plays an important role in mast cell-mediated cytotoxicity. Whereas lysates of rat peritoneal mast cells exhibited cytotoxicity toward WEHI-164, this was not found with lysates of unstimulated skin mast cells suggesting that TNF-alpha is not stored preformed in the latter. Killing of WEHI-164 cells by skin mast cells was enhanced by anti-IgE and there was a significant correlation between histamine release and cytotoxicity after activation with this stimulus. We conclude that human skin mast cells are a potential source of TNF-alpha and suggest that these cells, particularly after activation, might contribute to the synthesis of this multifunctional cytokine in inflammatory sites.     

Cytotoxicity of lymphoid cells induced by Maclura pomifera (MP) lectin.

The cytotoxic activity of lymphoid cells stimulated with Maclura pomifera (MP) lectin was investigated. Spleen cells of Lewis (LEW) or Brown Norway (BN) rats induced a cell-dependent release of ⁵¹Cr from syngeneic, allogeneic, and xenogeneic erythrocytes when incubated with MP for 4–16 hr. The activity of MP differed from that of concanavalin A (Con A). MP exhibited a greater activity with spleen cells while Con A was more active when bone marrow cells were tested. Activity induced by MP required the presence of the lectin for at least 4 hr and was inhibited by melibiose, an inhibitor of MP binding. MP also stimulated phagocytosis by peritoneal macrophages of LEW rats, but phagocytosis was not responsible for the cytotoxic effect measured by ⁵¹Cr release. The ability of aggressor cells to bind MP did not correlate with their cytotoxic activity. The cytotoxic activity of spleen cells from athymic nude mice was equivalent to that of cells from euthymic littermates when stimulated with MP.     

Mouse mannose-binding lectin-A and ficolin-A inhibit lipopolysaccharide-mediated pro-inflammatory responses on mast cells

It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response against bacterial lipopolysaccharide (LPS) stimulation. LPS-mediated pro-inflammatory cytokine productions on mBMMCs obtained from Toll-like receptor4 (TLR4)-deficient mice, TLR2-defficient mice, and their wildtype, were specifically attenuated by the addition of either mouse MBL-A or ficolin-A in a dose-dependent manner. However, the inhibitory effects by mouse MBL-A or ficolin-A were restored by the addition of mannose or N-acetylglucosamine, respectively. These results suggest that mouse MBL-A and ficolin-A bind to LPS via its carbohydrate-recognition domain and fibrinogen-like domain, respectively, whereby cytokine production by LPSmediated TLR4 in mBMMCs appears to be down-regulated, indicating that mouse MBL and ficolin may have an inhibitory function toward mouse TLR4-mediated excessive inflammation on the mast cells. [BMB Reports 2013; 46(7): 376-381]     

Antigenic properties of T cell antigen-specific receptors isolated from the surface of rabbit and mouse spleen and lymph node cells

The presence ofa allotypic determinants was tested in fractions obtained by gel filtration of antigen-specific receptors isolated by immunoadsorption from lymphoid cells of antigen-stimulateda3-3 rabbits. This technique, as well as the inhibition of the reaction of isolated receptors with anti-T cell receptor antisera (anti R) by anti-a3 antibodies failed to demonstrate the presence of a allotypie determinants. The inhibitory effect of antigen-specific receptors isolated from the lymphoid cells of stimulated A/J mice on the cytotoxic effect of anti-Ia antibodies on mouse spleen cells in the presence of rabbit complement was tested. All preparations inhibited the cytotoxic reaction with the average effectivity of 60%. In order to confirm the presence of Ia determinants on the rabbit and mouse T cell receptor molecules it was shown that the reactions of three anti-R antisera with 12 different receptor preparations were inhibited by anti-la antibodies. SDS-PAGE analyses of¹²⁵I-labelled mouse specific receptors and the precipitate obtained by anti-R antisera showed that T cell receptors were present in fractions with molar mass 100 and 85 kg/mol. The molar mass of the former fraction after reduction and alkylation was 45 kg/mol.     

Propagation of mouse cytotoxic clones with characteristics of natural killer (NK) cells

Splenocytes obtained from normal mice (BALB/c nude, BALB/c, C₃H, C57Bl/6) and from mice bearing lung or pulmonary carcinomas were propagated for 1–12 months in the presence of crude or mitogen-depleted T-cell growth factor (TCGF). Clones from several TCGF-propagated lymphoid cell lines were established by limiting dilution or the soft agar techniques. All the cultured lines and the majority of the clonal populations derived from them exhibited strong cytotoxic activity in vitro (⁵¹Cr release assay) toward a variety of syngeneic and allogeneic tumor target cells, both freshly obtained and passaged in culture, and both lymphoid and solid in origin, and including targets usually resistant to fresh NK cells. Considerable cytotoxic activity was also observed with several rat and human cultured tumor lines. Only low cytotoxic activity was detected against normal lymphoid mouse cells. Cloned populations generally exhibited more restricted target cytotoxicity than the parental cultured lines, and the pattern of reactivity varied among the clones. Of the clones tested for surface markers, all were positive for Thy 1.2, T200, and asialo GM1 and had strong binding to peanut agglutinin (PNA), all had undetectable receptors for IgG or IgM, and some were positive for Lyt 2. The cytotoxic activity was augmented by pretreatment of the effector cells with interferon and inhibited by the presence of mannose or galactose during the assay. Several clones were capable of mediating antibody-dependent cellular cytotoxicity and lectin-induced cellular cytotoxicity (LICC), and produced relatively large quantities of interferon and lymphotoxinlike material. The findings indicated continuous culturing in TCGF of previously antigen-nonstimulated mouse lymphocytes selects for the growth of at least two distinct populations with activated NK activity, one reacting preferentially with lymphoid tumor target cells (designated CNK-L), and the second reacting effectively with both lymphoid and solid tumor targets (designated CNK-SL). Both populations have several features of both T lymphocytes and NK cells.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

Presentation of IFN-gamma to nitric oxide-producing cells: a novel function for mast cells

We report that mast cells can bind and present IFN-gamma in a functionally active form to macrophages. Flow-cytometric analysis revealed that biotinylated IFN-gamma bound equally well to purified peritoneal mast cells from both IFN-gammaR knockout and wild-type mice, indicating a non-IFN-gammaR binding site. Purified peritoneal mast cells, loaded with IFN-gamma for 30 min and washed, were able to induce NO synthesis by peritoneal macrophages. This response required cell contact and expression of IFN-gammaR on the responding macrophages, but not the mast cells. Human HMC-1 mast cells were also able to present IFN-gamma to mouse macrophages. Enzyme treatment of mouse mast cells revealed that binding of IFN-gamma was predominantly to chondroitin sulfate B (dermatan sulfate). Binding of IFN-gamma to dermatan sulfate was confirmed by inhibition ELISA. This study demonstrates for the first time that mast cells can present IFN-gamma to other cells via glycosaminoglycans. Mast cells may act as a reservoir of surface-stored functionally active cytokines.     

Mast cells in a murine lung ischemia-reperfusion model of primary graft dysfunction

Primary graft dysfunction (PGD), as characterized by pulmonary infiltrates and high oxygen requirements shortly after reperfusion, is the major cause of early morbidity and mortality after lung transplantation. Donor, recipient and allograft-handling factors are thought to contribute, although new insights regarding pathogenesis are needed to guide approaches to prevention and therapy. Mast cells have been implicated in ischemic tissue injury in other model systems and in allograft rejection, leading to the hypothesis that mast cell degranulation contributes to lung injury following reperfusion injury.We tested this hypothesis in a mouse model of PGD involving reversible disruption of blood flow to one lung. Metrics of injury included albumin permeability, plasma extravasation, lung histopathology, and mast cell degranulation. Responses were assessed in wild-type (Kit^+/+) and mast cell-deficient (Kit^W-sh/W-sh) mice. Because mouse lungs have few mast cells compared with human lungs, we also tested responses in mice with lung mastocytosis generated by injecting bone marrow-derived cultured mast cells (BMCMC).We found that ischemic lung responses of mast cell-deficient Kit^W-sh/W-sh mice did not differ from those of Kit^+/+ mice, even after priming for injury using LPS. Degranulated mast cells were more abundant in ischemic than in non-ischemic BMCMC-injected Kit^W-sh/W-sh lungs. However, lung injury in BMCMC-injected Kit^W-sh/W-sh and Kit^+/+ mice did not differ in globally mast cell-deficient, uninjected Kit^W-sh/W-sh mice or in wild-type Kit^+/+ mice relatively deficient in lung mast cells.These findings predict that mast cells, although activated in lungs injured by ischemia and reperfusion, are not necessary for the development of PGD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0095-0) contains supplementary material, which is available to authorized users.     

Enhanced natural killer (NK) cell activity and NK-sensitive thymic cells in murine muscular dystrophy

Studies have shown that there is an abnormality in the thymus of dystrophic mice with respect to age-dependent thymus weight changes and altered morphology (T. DeKretser and B. Livett, Nature (London), 263, 682, 1976). Recently, others have shown that natural killer (NK) cells can lyse cells of a large, immature, rapidly dividing cell subpopulation within the thymus of normal young (3 weeks of age) mice (M. Hansson, K. Karre, R. Kiessling, J. Roder, B. Anderson, and P. Hayry, J. Immunol., 123, 765, 1979). The NK susceptibility of dystrophic mouse thymocytes as targets was therefore studied. Spleen cells from normal (+/+) and dystrophic ($\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$) male C57BL/6J mice 8–10 weeks old were passed over nylon wool and the nonadherent cells were incubated with ⁵¹Cr-labeled YAC-1 lymphoma target cells or thymocytes in a ⁵¹Cr-release assay. Spleen cells from dystrophic mice killed twofold more YAC-1 target cells than did spleen cells from normal mice. Thymocytes from 3- to 4-week-old dystrophic mice were three to four times more susceptible to NK lysis by dystrophic mouse spleen cells as compared with normal mouse spleen cells. Spleen cells from dystrophic mice had the same NK activity against dystrophic and normal mouse thymocytes as targets. Normal mouse spleen cells killed three- to fourfold more dystrophic mouse thymocytes than that of normal mouse thymocytes as targets. Target cellbinding studies revealed that conjugate-forming cells from nylon nonadherent dystrophic mouse spleen cells were found to be two- to fourfold greater than for normal mouse spleen cells using YAC-1 tumor cells as targets. The number of lymphocytes bound per YAC-1 target cell ranged from 2 to 5 for dystrophic mouse spleen cells as compared with 1 to 2 for the normal control group. Using both normal and dystrophic mouse thymocytes as targets, the conjugate-forming cells from dystrophic mouse spleen cells were also found to be twofold greater than in the normal control group. Cold target inhibition studies revealed that the natural killing of dystrophic mouse thymocytes was due to a YAC-1-reactive NK cell. Effector cell depletion studies using monoclonal anti-Thy-1.2 plus complement treatment and plastic petri dish adherence also revealed that the natural killing of dystrophic mouse thymocytes was not due to either T lymphocytes or macrophages. Taken together, these results show an increase in NK-sensitive thymocyte targets in dystrophic mice, in combination with an increase in splenic NK activity.     

Macrophage procoagulant-inducing activity of influenza-specific effector T cells

Three different types of immune mouse T cells raised against influenza virus were tested for their ability to induce the formation of macrophage procoagulant activity (MPCA) by a macrophage cell line PU5-1.8. They were primary spleen cells, taken 6 days after iv injection of virus, spleen cells from sensitized mice challenged with virus and cultured in vitro for 5 days (secondary cultured cells), and cloned T cells. With the last two preparations, some samples were K,D region restricted, Lyt 2⁺, and had cytotoxic activity; other samples were I region restricted, Lyt 2^?, and were not cytotoxic. Samples of a concanavalin A-activated T-cell supernatant which regularly induced MPCA with PU5-1.8 cells were included as controls in all assays. A few batches of T-cell preparations failed to induce MPCA production, however, most batches were active. Two sources of variation were detected: first, the number of cells (5-to 150-fold) needed to induce a certain level of MPCA, as measured by the decrease in clotting time; and second, the value of the gradient of the cell dose response. Both K,D- and I-region-restricted cells, either as cloned or secondary cultured cells, could induce MPCA but with the latter preparation, I-region-restricted cells were the better inducers by about eightfold. T cells tested in this way were also injected into mouse hind footpads and their ability to mediate delayed-type hypersensitivity (DTH) reactions was measured. A positive but not proportional correlation between the abilities to induce MPCA and mediate DTH activity for primary spleen cells was found, but this was not generally observed with cultured or cloned T cells.