IFN-gamma is not the only mediator of suppressed myelopoiesis produced by mononuclear cells from aplastic anemia patients

Peripheral blood mononuclear cells (PBMC) from patients with aplastic anemia (AA) and healthy donors were compared with regard to their ability to produce soluble factors with inhibitory activity on in vitro granulopoiesis (GM-CFC). Although PBMC from AA patients produced enhanced levels of IFN-gamma as compared to controls, this lymphokine was found not to be the main inhibitor of in vitro granulopoiesis. Other, non-IFN related factors were potent inhibitors of both the mature and the immature precursors for GM-CFC, could act across the species barrier and were of low molecular weight. Also PBMC from healthy donors produced a non-IFN mediated GM-CFC inhibitory factor, but to a lesser degree and acting only on one type of myeloid precursors. The possible implications of these findings in relation to the etiology of AA will be discussed.     

Pretreatment with granulocyte colony-stimulating factor reduces myelopoiesis in irradiated mice

The purpose of this study was to investigate effects of the treatment prior to irradiation with granulocyte colony-stimulating factor (G-CSF) on hematopoiesis in B10CBAF1 mice exposed to a sublethal dose of 6.5 Gy of 60Co gamma radiation. G-CSF was administered in a 4-day regimen (3 microg/day); irradiation followed 3 h after the last injection of G-CSF. Such a treatment was found to stimulate granulopoiesis, as shown by increased counts of granulocyte-macrophage progenitor cells (GM-CFC) and of granulocytic cells in the femoral marrow and spleen at the time of irradiation. However, postirradiation counts of GM-CFC and granulocytic cells in the marrow of mice pretreated with G-CSF were reduced up to day 18 after irradiation. Interestingly, the D0 values for marrow GM-CFC determined 1 h after in vivo irradiation were 1.98 Gy for controls and 2.47 Gy for mice pretreated with G-CSF, indicating a decreased radiosensitivity of these cells after drug treatment. The inhibitory effects of the pretreatment with G-CSF on the postirradiation granulopoiesis could be attributed to the phenomenon of "rebound quiescence" which can occur after cessation of the treatment with growth factors. Postirradiation recovery of erythropoiesis in the spleen of mice pretreated with G-CSF exhibited a dramatic increase and compensated for the decreased erythropoiesis in the marrow at the time of irradiation. This complexity of the hematopoietic response should be taken into account when administering G-CSF in preirradiation regimens.     

Regulation of human natural cytotoxicity by IgG. III. Interaction between negative regulation by monomeric IgG and positive regulation by interferons of different types

Our prior reported results have demonstrated the dose-dependent inhibition of human natural killer (NK) cell activity upon treatment of peripheral blood mononuclear cells (PBMC) with monomeric IgG (mIgG) prior to the cytotoxic assay. In the present study, the combined effects on NK activity of human interferon (IFN) of each of the three types and mIgG, respectively, were determined. NK cells incubated with IFN alpha or IFN beta had augmented cytotoxicity against K562 target cells but remained responsive to negative regulation by mIgG. PBMC treated with human recombinant IFN gamma had unchanged cytotoxic activity but became partially resistant to suppression by mIgG. This ability of IFN gamma to interfere with the negative regulation of NK activity by cytophilic mIgG was seen when the cytokine was preincubated with effector cells prior to, simultaneously with, or after their exposure to inhibitor protein. These data provide some clues regarding the possible biological significance of the mIgG-induced down-regulation of NK cells which, when required for host protection, might be appreciably reversed or blocked by IFN gamma produced by NK cells or T cells in response to various agents.     

Autologous enhancement by interferon of natural killer activity of human peripheral blood lymphocytes

The in vitro action of interferon (IFN)-alpha and IFN-gamma from six healthy donors and ten patients with multiple sclerosis (MS) on natural killer (INK) activity of peripheral blood lymphocytes (PBL) was studied in an autologous system. The NK activity of PBL was detected by a cytotoxic test using (3)H-uridine human erythromyeloblast K562 cells. Autologous IFN-alpha and IFN-gamma did not augment NK activity of PBL from healthy donors in vitro, whereas in samples from MS patients the IFNs strongly stimulated NK cell cytotoxic function. This stimulation suggests the existence of an inhibitor of regulatory IFN action, that is produced in healthy donors simultaneously with IFN in response to IFN induction, but which is lacking in commercial IFN preparations. The factor-containing supernatants from healthy donors reduced the stimulatory action of autologous IFNs in patients with MS almost until complete blockade. Because this inhibitor was absent in patients with MS, deficiency of an inhibitor of IFN regulatory action in MS could open the way to treatment of this compartment of the immune system.     

IFN-gamma treatment of K562 cells inhibits natural killer cell triggering and decreases the susceptibility to lysis by cytoplasmic granules from large granular lymphocytes

Pretreatment of human K562 leukemia cells with rIFN-alpha and rIFN-gamma resulted in decreased susceptibility to lysis by human peripheral blood NK cells. The reduction of NK-susceptibility after IFN treatment was not due to a general effect of IFN on the stability of the cell membrane because the susceptibility of K562 cells to lysis by antibodies plus C, distilled water, or lysolecithin was unaffected. Binding studies with effector cell preparations enriched for NK cells with large granular lymphocyte morphology revealed no difference in binding to control and IFN-gamma-treated target cells. The sensitivity to soluble NK cytotoxic factors was not affected significantly by the IFN treatment. In contrast, the susceptibility of IFN-treated target cells to the cytotoxic activity of purified cytoplasmic granules from a rat large granular lymphocyte tumor was significantly reduced, indicating that the IFN-induced resistance acted at the level of susceptibility to the lytic mechanism of NK cells. However, IFN-alpha was more effective than IFN-gamma in inducing resistance to the cytoplasmic granules although resulting in only a weak resistance in the cell-mediated cytotoxic assay. IFN-gamma but not IFN-alpha caused a reduction in the frequency of effector cells that had reoriented their Golgi apparatus toward their bound target cell. In addition, IFN-gamma treated K562 cells failed to elicit an influx of Ca2+ into effector cells. Taken together, the results suggest that IFN-gamma in addition to an increased resistance to the lytic molecules released by NK cells can also induce changes in the target cells which prevent the triggering and activation of the effector cell.     

Histamine H2-receptor-mediated regulation of human natural killer cell activity

We have investigated effects of histamine on the spontaneous cytotoxic activity of human natural killer (NK) cells in vitro. Addition of histamine (10(-3) to 10(-7) M) to assay cultures of Percoll-fractionated mononuclear cells (MNC) and erythroleukemic K 562 target cells resulted in a strong enhancement of the cytotoxicity of low-density MNC, enriched for NK cell cytotoxicity (NKCC). No enhancing or suppressing effects of histamine could be detected after removal of monocytes/adherent cells from the effector cell suspensions. When unfractionated MNC were used as NK effectors, similar results were obtained, i.e., dose-dependent enhancement of NKCC by histamine in the presence of monocytes and lack of effect in nonadherent effector cells. Freshly isolated monocytes displayed low spontaneous cytotoxicity against K 562 targets and were not induced by histamine. The histamine-induced enhancement was mimicked by dimaprit, a specific histamine H2-receptor agonist, but not by N-methyldimaprit, a chemical control for H2-receptor agonist activity of dimaprit. Furthermore, the enhancement was completely antagonized by the specific histamine H2-receptor antagonists cimetidine and ranitidine. The effect of histamine could not be ascribed to endogenous interferon (IFN) production, since no IFN activity could be detected in histamine-treated MNC effectors. Also, the enhancing effects of histamine and human leukocyte IFN-alpha were clearly additive. On the basis of these findings, we suggest that histamine, via specific activation of H2 receptors, may be an important regulator of human NK cell activity.     

IFN-beta 2/IL-6 augments the activity of human natural killer cells

MHC nonrestricted cytotoxic cells play an important role in the killing of tumor cells in vitro and potentially in vivo. The activity of these cells is regulated by several cytokines such as IL-2 and IFN. In the present study we provide first evidence that IL-6 significantly augments the cytotoxic activity of human NK cells. IL-6 is produced by many different cells and is also known as IFN-beta 2, B cell stimulatory factor 2, hybridoma growth factor, hepatocyte-stimulating factor, and 26 kDa protein. IL-6 stimulates the activity of human CD3- NK cells but not that of CD3+ non-MHC-restricted cytotoxic T lymphocytes. As is the case with IL-2, the IL-6-mediated augmented cytotoxicity was a result of a more efficient lysis, but was not caused by an increased effector to target cell binding. Moreover, the effect of IL-6 on NK cell activity was blocked by a mAb directed against IL-2, and IL-6 itself was found to be a potent inducer of IL-2 production in cultured human PBMC. Thus it may be concluded that IL-6 enhances the cytotoxic activity of NK cells via IL-2. This newly recognized property of IL-6, which is produced by almost any cell, may be of importance in host defense against microbes and malignancies and therefore could contribute to improve the adoptive immunotherapy by using lymphokine-activated killer cells.     

The role of the thymus in the maintenance of natural killer cells in vivo

This report describes a model for investigating the role of the thymus in regulating natural killer (NK) cell activity in vivo. Evidence is presented that the thymus can regulate NK cells, and that at least some NK cells can develop without thymic help. Marrow from thymectomized rats depleted of circulating T cells by thoracic duct cannulation was transplanted into rats without a thymus (1 degree ATX.BM). These 1 degree ATX.BM rats had NK cell levels above controls 3 months after reconstitution but markedly depressed NK cell levels by 9 months. When 1 degree ATX.BM marrow was used to reconstitute rats with or without a thymus, those without a thymus (2 degrees ATX.BM) exhibited low NK cell levels after 3 months, and a similar result was obtained when 2 degrees ATX.BM marrow was used to reconstitute 3 degrees ATX.BM rats. The low NK cell levels in 2 degrees and 3 degrees ATX.BM rats were due to a deficiency in spontaneously cytotoxic NK cells, as they had normal numbers of interferon-responsive pre-NK cells. Spleen cells from 2 degrees and 3 degrees ATX.BM rats produced less interferon than control spleen cells when cultured with P815 tumor cells in vitro. However, 2 degrees and 3 degrees ATX.BM rats had higher numbers of large granular lymphocytes than controls despite their low NK cell levels. In marked contrast to 2 degrees and 3 degrees ATX.BM rats, spleen cells from 4 degrees ATX.BM rats had higher levels of cytotoxicity and a higher frequency of both spontaneously cytotoxic and pre-NK cells than controls. The 4 degrees ATX.BM rats also had the highest frequency of large granular lymphocytes in the spleen.     

Interferon gamma encapsulated into liposomes enhances the activity of monocytes and natural killer cells and has antiproliferative effects on tumor cells in vitro

The effects of human interferon gamma (IFN gamma) encapsulated into liposomes were investigated in vitro. Monocytes were induced to release a cytotoxic factor with either IFN gamma encapsulated into liposomes, free IFN gamma or lipopolysaccharide (LPS). If IFN gamma was applied in the liposomal form, less IFN activity was required to stimulate monocytes. Most of the cytotoxic factor was secreted during the first 4 h of stimulation. The cytotoxic factor in supernatants from PMNLs was completely neutralized by a monospecific polyclonal antiserum to tumor necrosis factor (TNF). Combining subthreshold doses of IFN gamma liposomes or IFN gamma with lipopolysaccharide synergistically enhanced the release of TNF. In fluorescence analysis, altered expression of the class II HLA-DR antigen on LeuM3 positive monocytes was induced with IFN gamma liposomes as well as with IFN gamma. Not only monocytes but also natural killer (NK) cells were stimulated to higher cytotoxicity by IFN gamma liposomes in a dose-dependent manner. In comparison with IFN gamma, the same amount of activity was necessary for adequate stimulation of NK-cells against the K562 target cells. Furthermore, the antiproliferative effects of IFN gamma liposomes and free IFN gamma on several human tumor cell lines was compared. Among several cell lines tested, U937 and A549 turned out to be sensitive to IFN gamma, and both cell lines reacted with 50% growth inhibition at a lower amount of gamma presented by liposomes than in the free form. These data show production of IFN gamma liposomes which possess immunomodulatory and antiproliferative activity in vitro. In several of the test systems studied, liposome-encapsulated IFN gamma was more effective than free IFN gamma.     

Interleukin 2 inhibits in vitro granulocyte-macrophage colony formation

We have previously shown that murine bone marrow cells cultured with interleukin 2 (IL-2) produce interferon-alpha/beta (MuIFN-alpha/beta) and that IFN-alpha/beta can suppress in vitro granulocyte-macrophage colony-forming cell formation (GM-CFC). In this study, IL-2 was directly assessed for its ability to inhibit in vitro granulocyte and/or macrophage colony-forming cell formation (GM-CFC/M-CFC). C57BL/6 bone marrow cells were cultured with different colony-stimulating factors (CSF), i.e., partially purified macrophage-CSF (M-CSF) or recombinant granulocyte and macrophage CSF (GM-CSF) in the presence or absence of different IL-2 preparations. Partially purified mouse IL-2 or recombinant human or mouse IL-2 (rHuIL-2 and rMuIL-2) totally inhibit GM-CFC and M-CFC formation at 7 days of culture. The level of inhibition mediated by IL-2 was concentration-dependent, with as little as 1 U/ml giving total inhibition of colony formation. The ability of IL-2 to inhibit colony formation was completely abolished by treatment with antisera to IL-2. MuIFN-alpha/beta and MuIFN-gamma appeared to play no role in IL-2-induced myelo-suppression in that addition of antisera to these IFN failed to block IL-2-induced suppression. Myelo-suppression mediated by IL-2 was independent of the concentration of CSF used in the bone marrow cultures. Suppression was also not dependent upon the initial presence of T cells or natural killer (NK) cells. Bone marrow cells depleted of Thy-1+, Lyt-1+, Lyt-2+, NK-1.1+, Asialo GM1+, or Qa-5+ cells were as susceptible to IL-2 induced suppression as untreated or complement-treated bone marrow cells. These results suggest that IL-2 may play an important role in regulating different aspects of hematopoiesis.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

Inhibition of mouse bone marrow granulocyte-macrophage colony formation by factors derived from natural killer cells: an in vitro model for hybrid resistance

Investigations were performed to study whether soluble factors produced by NK-cells could mediate "hybrid resistance" in vitro. NK-cells enriched from spleens of B6D2F1 hybrid mice were incubated with parental B6 bone marrow, and the effect of the derived supernatants on the development of granulocyte-macrophage colony forming cells (GM-CFC) was assessed. Cell free supernatants obtained from low density cells (LDC) of B6D2F1 hybrids stimulated with bone marrow cells (BMC) from B6 mice inhibited GM-CFC formation. The inhibition was similar using B6, D2 or B6D2F1 bone marrow cells as the targets for GM-CFC growth. Our findings suggest that NK cells from F1 hybrid mice when stimulated with BMC from B6 mice release inhibitory factors, different from IFN-gamma and that this production may represent a mechanism of natural resistance to parental H-2b bone marrow grafts.     

Opposite effects of serotonin and interferon-α on the membrane potential and function of human natural killer cells

Summary In an earlier article, we reported that serotonin (5-hydroxytryptamine, 5-HT) inhibits the natural killer cell (NK) cytotoxicity of human whole blood in a dose-dependent manner and that natural human interferon-α (IFN-α) partially eliminates this effect. Because natural IFN-α might contain factors other than IFN, we repeated these experiments with recombinant human interferon-α (rhIFN-α) and separated blood lymphocytes enriched with NK cells and then demonstrated that IFN really is responsible for this effect. Furthermore, this investigation was carried out to clarify the mechanisms of the action of 5-HT and of rhIFN-α on NK cells. The inhibition of the cytotoxicity was pronounced when 5-HT was added at the onset of the cytotoxic assay, whereas the pretreatment of lymphocytes for 18 h only led to a slight inhibition. Moreover, rhIFN-α applied 1 h before or 1 h after the addition of 5-HT decreased the inhibitory effect of 5-HT. Flow cytometric analysis involving the use of a voltage-sensitive dye, oxonol, revealed that 5-HT depolarized, whereas rhIFN-α hyperpolarized the plasma membrane of the lymphocytes. Thus, it seems likely that the inhibitory effect of 5-HT on the cytotoxicity of peripheral human lymphocytes is due to the depolarization on the plasma membrane of the effector cells and that rhIFN-α antagonizes this ability via its hyperpolarizing activity.     

Effect of the in vivo application of granulocyte colony‐stimulating factor on NK cells in bone marrow and peripheral blood

Granulocyte colony‐stimulating factor (G‐CSF) has been widely used in the field of allogeneic haematopoietic stem cell transplantation (allo‐HSCT) for priming donor stem cells from the bone marrow (BM) to peripheral blood (PB) to collect stem cells more conveniently. Donor‐derived natural killer (NK) cells have important antitumour functions and immune regulatory roles post‐allo‐HSCT. The aim of this study was to evaluate the effect of G‐CSF on donors' NK cells in BM and PB. The percentage of NK cells among nuclear cells and lymphocyte was significantly decreased and led to increased ratio of T and NK cells in BM and PB post‐G‐CSF in vivo application. Relative expansion of CD56^bri NK cells led to a decreased ratio of CD56^dim and CD56^bri NK subsets in BM and PB post‐G‐CSF in vivo application. The expression of CD62L, CD54, CD94, NKP30 and CXCR4 on NK cells was significantly increased in PB after G‐CSF treatment. G‐CSF treatment decreased the IFN‐γ‐secreting NK population (NK1) dramatically in BM and PB, but increased the IL‐13‐secreting NK (NK2), TGF‐β‐secreting NK (NK3) and IL‐10‐secreting NK (NKr) populations significantly in BM. Clinical data demonstrated that higher doses of NK1 infused into the allograft correlated with an increased incidence of chronic graft‐vs‐host disease post‐transplantation. Taken together, our results show that the in vivo application of G‐CSF can modulate NK subpopulations, leading to an increased ratio of T and NK cells and decreased ratio of CD56^dim and CD56^bri NK cells as well as decreased NK1 populations in both PB and BM.     

Effects of diethyldithiocarbamate, an inhibitor of interferon antiviral activity, upon human natural killer cells

In support of a postulated role of the Cu++-dependent enzyme, superoxide dismutase (SOD), in antiviral effects of interferon (IFN), a close correspondence was previously shown to exist between inactivation of cellular SOD and concomitant blockade of IFN antiviral activity in fibroblasts by the Cu++-chelating agent, diethyldithiocarbamate (DDC). To further define the extent of "anti-IFN" activity, we initiated studies of DDC effects on IFN stimulation in the NK cell system. Unexpectedly, DDC directly inhibited cytotoxicity mediated by unstimulated NK cells. Pronounced inactivation occurred rapidly (less than 30 min), but was spontaneously reversible in the absence of DDC. Neither cell viability nor lymphocyte binding to target cells was detectably affected. Preincubation of DDC with Cu++ or Zn++ failed to neutralize its inhibitory effects nor could function be restored in DDC-pretreated NK cells by subsequent addition of Cu++, Zn++, Mg++, or Ca++. DDC treatment that inactivated NK cells did not detectably alter lymphocyte SOD activity. Thus, inhibition was probably not attributable to chelating properties of DDC. N-ethyl maleimide (NEM) and para-( hydroxymercuri ) benzoic acid ( PMBA ), enzyme inhibitors that preferentially react with sulfhydryl groups, both inactivated NK cells in a time- and dose-dependent manner similar to that of DDC. Preincubation with the sulfhydryl compound, cysteine, neutralized in parallel fashion the capacity of NEM, PMBA , and DDC to inhibit NK cell activity. Thus, a previously unreported reactivity of DDC with sulfhydryl groups appeared to be the basis of inhibition. NK cells incubated 1 hr with IFN and subsequently cultured 17 to 23 hr without IFN were activated to an extent comparable to cells continuously incubated 18 to 24 hr with IFN. Exposure to IFN for 1 hr was therefore sufficient to commit NK cells to acquisition of a fully activated state. Whether preactivated by a 1-hr or 18- to 24-hr IFN treatment, activated NK cells retained the DDC-sensitive phenotype characteristic of fresh unstimulated NK cells. Thus, prolonged IFN treatment did not render NK cells resistant to DDC or preferentially activate a DDC-sensitive NK cell subset. An 18- to 24-hr incubation of DDC-pretreated cells in the continual presence of IFN resulted in the boosting of NK cell activity. However, the 1-hr IFN pulse treatment protocol was consistently ineffective in boosting when IFN was added just after DDC-pretreatment. These results strongly suggested that DDC temporarily rendered NK cells unresponsive to what, under normal circumstances, approximated an optimally potentiating IFN stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of postbinding target cell lysis and of lymphokine-induced enhancement of human natural killer cell activity by in vitro exposure to ultraviolet B radiation

In vitro exposure of human peripheral blood mononuclear cells (PBMC) to ultraviolet B (uvB) radiation has been shown to inhibit natural killer (NK) cell-mediated cytotoxicity in a dose-dependent fashion. The purpose of this study was to examine the manner by which uvB produced these deleterious effects. Inhibition of NK activity was not due to lethal injury to NK cells since the viability of cell populations enriched for NK activity was greater than 90% with the uvB doses employed. uvB appeared to directly affect NK cells since procedures which removed suppressor mechanisms, such as removal of monocytes and pharmacologic inhibition of the cyclooxygenase pathway, failed to reverse the response. Furthermore, no suppression of activity of unirradiated NK cells could be produced by coincubation of unirradiated NK cells with uv-irradiated NK cells. When the single cell assay for binding and killing was employed to determine at which stage in the lytic sequence inhibition occurred, it was found that binding was normal but lysis of bound targets and the recycling capacity of active NK cells were markedly reduced. At uvB doses above 50 J/m2, both interferon alpha (IFN-alpha) and interleukin 2 (IL-2) were ineffective in augmenting NK cell-mediated cytotoxic reactions after cells had been irradiated with uvB. Furthermore, incubation of NK cells with IFN-alpha prior to irradiation failed to protect against the inhibitory effects. These studies provide evidence that in vitro exposure of NK cells to uvB radiation inhibits their function by a direct nonlethal effect and that this inhibition occurs selectively at the postbinding stage of target cell lysis.     

Role of interferon in human natural killer activity against target cells infected with HSV-1

Human natural killer (NK) cells show high cytotoxic activity against target cells infected with herpes simplex virus type 1 (HSV-1). Substantial amounts of interferon (IFN) were generated in co-cultures of NK effector cells and infected target cells; however, the cytotoxic activity seen against a specific infected cell target did not correlate with the amount of IFN induced. The production of IFN increased steadily from 4 to 18 hr of co-culture, as did NK activity; however, IFN production peaked 4 hr later than NK activity. Pretreatment of NK effector cells with exogenous IFN increased cytotoxic activity against all targets tested, but the differential pattern of reactivity against cells infected with wild type and mutant viruses was unaltered. When effector cells were treated with the RNA synthesis inhibitor actinomycin D before co-culture with virus-infected targets, IFN production was markedly reduced, without a concomitant reduction in cytotoxicity. Similarly, the addition of anti-IFN antiserum to co-cultures greatly decreased the available IFN present, but had no effect on NK activity. We conclude that the induction of cytotoxic activity in co-cultures of NK effector cells and HSV-1-infected target cells is independent of the induction of IFN.     

Mechanism of inhibition of human natural killer activity by ultraviolet radiation

Ultraviolet radiation (UVR) has been shown to inhibit various immune functions in vivo and in vitro. We have confirmed that UVR inhibits human natural killer (NK) activity in vitro and have shown that UVR inhibits human ADCC. In this report, the mechanism by which UVR inhibits NK function was investigated by analyzing the stage at which the inhibiting activity occurs and the ability of the NK cells to release cytotoxic factors previously shown to be involved in CMC. Single cell assays in Agarose revealed that inhibition of NK activity was localized at the postbinding lethal hit stage rather than the initial recognition or binding stage of lysis. We then examined whether UV-treated cells were able to release cytotoxic factors after stimulation with target cells. As expected, stimulated cells released cytotoxic factors, yet, surprisingly, these factors were also released in the absence of stimulator cells. The spontaneous release was detectable in the supernatants as early as 30 min after UV irradiation. The lytic material examined in 48- to 72-hr viability assays was not NK specific, because lysis was obtained with a wide range of NK sensitive and resistant target cells. These results demonstrate that UVR does not alter the capacity of the cells to secrete cytotoxic material, but in fact enhances its release. Several possible mechanisms are proposed to explain the UVR-induced NK inhibition.     

The inhibitory receptor IRp60 (CD300a) is expressed and functional on human mast cells

Mast cell-mediated responses are likely to be regulated by the cross talk between activatory and inhibitory signals. We have screened human cord blood mast cells for recently characterized inhibitory receptors expressed on NK cells. We found that IRp60, an Ig superfamily member, is expressed on human mast cells. On NK cells, IRp60 cross-linking leads to the inhibition of cytotoxic activity vs target cells in vitro. IRp60 is constitutively expressed on mast cells but is down-regulated in vitro by the eosinophil proteins major basic protein and eosinophil-derived neurotoxin. An immune complex-mediated cross-linking of IRp60 led to inhibition of IgE-induced degranulation and stem cell factor-mediated survival via a mechanism involving tyrosine phosphorylation, phosphatase recruitment, and termination of cellular calcium influx. To evaluate the role of IRp60 in regulation of allergic responses in vivo, a murine model of allergic peritonitis was used in which the murine homolog of IRp60, LMIR1, was neutralized in BALB/c mice by mAbs. This neutralization led to a significantly augmented release of inflammatory mediators and eosinophilic infiltration. These data demonstrate a novel pathway for the regulation of human mast cell function and allergic responses, indicating IRp60 as a candidate target for future treatment of allergic and mast cell-associated diseases.