GM-CSF and IL-4 are not involved in IVIG-mediated amelioration of ITP in mice: a role for IL-11 cannot be ruled out.

Previously, we have reported that interleukin (IL)-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-11, but not IL-33, are up-regulated in two strains of mice with immune thrombocytopenia (ITP) that are responsive to intravenous immunoglobulin (IVIg) treatment. Previously, IL-4 was ruled out in the mechanism of IVIg; however, other publications have suggested this cytokine as a major player in the mechanism of IVIg action. Thus, we sought to further investigate a role for IL-4 and, in addition, GM-CSF and IL-11 in the mechanism of action of IVIg using a murine model of ITP. A passive platelet antibody model was used to generate ITP in IL-4 receptor knock-out (IL-4R-/- ), IL-11 receptor knock-out (IL-11Rα-/- ) and GM-CSF knock-out (Csf2-/- ) mice. We also used a neutralizing antibody to IL-11 and recombinant human IL-11 (rhIL-11) in addition to depleting basophils in vivo to study the effect of IVIg to ameliorate ITP. Our results showed that basophils, IL-4 and GM-CSF were unimportant in both ITP induction and its amelioration by IVIg. The role of IL-11 in these processes was less clear. Even though IL-11Rα-/- mice with ITP responded to IVIg similarly to wild-type (WT) mice, treatment of ITP WT mice with rhIL-11 instead of IVIg showed an increase in platelet numbers and WT mice administered anti-IL-11 showed a significant reduction in the ability of IVIg to ameliorate the ITP. Our findings indicate that neither IL-4, basophils or GM-CSF have roles in IVIg amelioration of ITP; however, a role for IL-11 requires further study.

7th International Immunoglobulin Conference: Mechanisms of action.

Although intravenous immunoglobulin (IVIg) is widely used for replacement therapy in immunodeficiencies and to treat autoimmune and inflammatory diseases, its mechanisms of action are not fully understood. Examination of immunoglobulin (Ig) receptors, including the Fc-gamma receptors (FCγRs) and the neonatal Fc receptor, have revealed genetic variations that are linked to autoimmune diseases and to the efficacy of IVIg treatment. However, the beneficial effect of IVIg encompasses multiple mechanisms of action. One of these is scavenging of activated complement fragments, such as C3a, C5a, C3b and C4b, by infused Ig molecules. This interaction prevents binding of complement fragments to their receptors on target cells, thus attenuating the immune damage. Additionally, anti-inflammatory effects may be facilitated by IgA via specific receptors and/or complement scavenging. Glycosylation of both the Fc- and Fab-fragments has also been implicated in the anti-inflammatory action of IVIg. Although there is evidence to support a role for sialylated IgG glycovariants in mediating the effect of IVIg, evidence from animal models of inflammatory disease suggest that sialylation may not be a critical factor. However, an increase in IgG glycosylation has been observed following IVIg treatment in Guillain-Barré syndrome patients, and this has been associated with improved clinical outcomes.

7th International Immunoglobulin Conference: Mechanisms of action.

The mechanism of action by which therapeutic administration of intravenous immunoglobulin (IVIg) is able to provide a beneficial effect in autoimmune and inflammatory diseases is not yet fully understood, but current research is providing some answers. Signalling via receptors that interact with immunoglobulin (Ig) is crucial, and genetic polymorphisms of the Fc receptors have clear links to disease and also appear to influence the outcome of IVIg treatment. Glycosylation of the IgG, Fc- or Fab-fragments has a role in enhancing or blocking the pro- and anti-inflammatory effector functions. In addition, and independently of Fc receptors and glycosylation, Fc fragment and the constant domain of the Fab fragment contain binding sites for activated complement fragments that mediate complement-scavenging based immunomodulation. Although IgG Fc sialylation may not be critical for IVIg activity, research in some diseases suggests that it is associated with improved clinical outcomes. Therefore, further investigation of how IgG and IgA receptor expression and regulation affects the outcome of IVIg treatment may further clarify the mechanisms behind IVIg, and provide valuable guidance for future treatment paradigms.

The medium is the message: glycosphingolipids and their soluble analogues.

We have made adamantylGSLs by substituting the fatty acids of primarily, globotriaosyl ceramide(Gb(3)) and sulfogalactosyl ceramide(SGC), with the rigid alpha-adamantane hydrocarbon frame. These analogues have proven to be remarkably water-soluble but retain the receptor function of the parent membrane GSL. AdaGb(3) prevents the binding of verotoxins to target cells but increased pathology in vivo, likely due to the partitioning into receptor negative target cells to provide pseudo-receptors. Preincubation of HIV with adaGb(3) prevents cellular infection in vitro and viral-host cell fusion. Cellular accumulation of Gb(3) reduces HIV susceptibility in vitro, whereas lack of Gb(3) promotes infection, suggesting that Gb(3) expression could be a novel risk factor for HIV susceptibility. AdaGb(3) has proven to be a new inhibitor for the MDR1 drug pump (P-glycoprotein) and can reverse drug resistance in cell culture. AdaSGC is bound by hsp70/hsc70 within the N-terminal ATPase domain and inhibits chaperone function. When added to cells transfected with the DeltaF508 CFTR mutant, adaSGC was able to decrease ER degradation of this mutant protein, an hsc70 dependent process. Our finding that DeltaF508 CFTR expressing cells show reduced SGC biosynthesis suggests that SGC could be an additional natural regulator of the hsp70 chaperone ATPase cycle.

c-Src tyrosine kinase co-associates with and phosphorylates signal transducer and activator of transcription 5b which mediates the proliferation of normal human B lymphocytes.

c-Src is the normal human cellular protein homologue of the viral oncogene v-src. c-Src activity was reported recently to increase in CD40-activated human B lymphocytes, suggesting its involvement in proliferation. To elucidate the exact role of c-Src in this process, we investigated the effects of c-Src over-expression on normal B lymphocyte growth. B lymphocytes purified from human peripheral blood were infected with Ad5/F35 vector encoding either a constitutively active c-Src (c-Src/dominant-positive) or a dominant-negative c-Src (c-Src/DN). Little variation of B lymphocytes expansion could be observed between control enhanced yellow fluorescent protein and c-Src/dominant-positive-infected cells. In contrast, over-expression of c-Src/DN results in a 40% inhibition of B lymphocyte expansion. These results suggest that DN c-Src may compete with endogenous c-Src, resulting in partial inhibition of a transcriptional pathway involved in B lymphocyte proliferation. We demonstrate further that c-Src can phosphorylate signal transducer and activator of transcription 5b (STAT5b) on tyrosine 699 and that c-Src and STAT5b co-associate during B lymphocyte proliferation. These results confirm an important role for c-Src in the expansion of normal human B lymphocytes in vitro, in which c-Src may regulate STAT5b in the intracellular signalling pathway important for the proliferation of normal human B lymphocytes.

Use of modified U1 snRNAs to inhibit HIV-1 replication.

Control of RNA processing plays a central role in regulating the replication of HIV-1, in particular the 3' polyadenylation of viral RNA. Based on the demonstration that polyadenylation of mRNAs can be disrupted by the targeted binding of modified U1 snRNA, we examined whether binding of U1 snRNAs to conserved 10 nt regions within the terminal exon of HIV-1 was able to inhibit viral structural protein expression. In this report, we demonstrate that U1 snRNAs complementary to 5 of the 15 regions targeted result in significant suppression of HIV-1 protein expression and viral replication coincident with loss of viral RNA. Suppression of viral gene expression is dependent upon appropriate assembly of a U1 snRNP particle as mutations of U1 snRNA that affect binding of U1 70K or Sm proteins significantly reduced efficacy. However, constructs lacking U1A binding sites retained significant anti-viral activity. This finding suggests a role for these mutants in situations where the wild-type constructs cause toxic effects. The conserved nature of the sequences targeted and the high efficacy of the constructs suggests that this strategy has significant potential as an HIV therapeutic.

Irradiated KHYG-1 retains cytotoxicity: potential for adoptive immunotherapy with a natural killer cell line.

PURPOSE: To evaluate gamma-irradiation on KHYG-1, a highly cytotoxic natural killer (NK) cell line and potential candidate for cancer immunotherapy. METHODS AND MATERIALS: The NK cell line KHYG-1 was irradiated at 1 gray (Gy) to 50 Gy with gamma-irradiation, and evaluated for cell proliferation, cell survival, and cytotoxicity against tumor targets. RESULTS: We showed that a dose of at least 10 Gy was sufficient to inhibit proliferation of KHYG-1 within the first day but not its cytolytic activity. While 50 Gy had an apoptotic effect in the first hours after irradiation, the killing of K562 and HL60 targets was not different from non-irradiated cells but was reduced for the Ph + myeloid leukemia lines, EM-2 and EM-3. CONCLUSIONS: gamma-irradiation (at least 10 Gy) of KHYG-1 inhibits cell proliferation but does not diminish its enhanced cytolytic activity against several tumor targets. This study suggests that KHYG-1 may be a feasible immunotherapeutic agent in the treatment of cancers.

Chloride-channel block inhibits T lymphocyte activation and signalling.

Both large- and small-conductance chloride (Cl-) channels have been found in human T lymphocytes; however, apart from possible roles in mediating regulatory volume decrease, their functions are not understood. We have used patch-clamp electrophysiology, Ca2+ spectrofluorometry, and Western blot assay for phosphotyrosine to investigate the effects of blocking Cl- channels on proliferation and on specific events in the activation of normal human T cells. Four chemically distinct Cl- channel blockers inhibited both the small-conductance Cl- channels and phytohemagglutinin (PHA)-induced lymphocyte proliferation in a similar dose-dependent manner; their order of potency was 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB) > 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS) > flufenamic acid >> IAA-94. The Cl- channel blockers inhibited both the PHA-induced mobilization of Ca2+ and the rapid tyrosine phosphorylation of several polypeptides. Cell proliferation was not rescued by the Ca+ ionophore ionomycin or by addition of exogenous interleukin-2 (IL-2). Moreover, the blockers also inhibited phosphotyrosine expression in IL-2-treated, activated lymphoblasts. Thus, our results support a role for Cl- channels in early, PHA-evoked signalling and in later, II-2-dependent stages of lymphocyte activation and proliferation.

Analysis of the synergistic stimulation of mouse macrophage proliferation by macrophage colony-stimulating factor (CSF-1) and tumor necrosis factor alpha (TNF-alpha).

Tumor necrosis factor alpha (TNF-alpha) more than doubles tritiated thymidine ([3H]TdR) uptake in mouse macrophages stimulated by macrophage colony-stimulating factor (CSF-1). However, nothing is known of how TNF-alpha affects this increase or even whether it is manifested by increased cellular proliferation. Here we characterize the effects of TNF-alpha on CSF-1-stimulated proliferation of both primary cells (bone marrow-derived macrophages, BMMs) and a cloned growth factor-dependent macrophage cell line (S1). We show that the TNF-alpha-induced increase in [3H]TdR uptake of CSF-1-stimulated macrophages is directly proportional to an increase in the DNA content of the culture and that the effects of TNF-alpha are direct and independent of cell number. TNF-alpha decreases the population doubling time of log-phase growing macrophages having quite different growth rates to the same (approximately 30%) extent: the doubling time of BMMs decreases from 24 to 17 h and that of S1 cells from 17 to 13 h. TNF-alpha exerts its effects on log-phase growth by increasing to the same proportion CSF-1-stimulated proliferation at all concentrations of CSF-1; that is, TNF-alpha does not shift, but rather amplifies, the CSF-1 dose-response curve. Although TNF-alpha alone does not stimulate macrophage proliferation, its presence in S1 cell cultures coming to quiescence after withdrawal of CSF-1 greatly increases subsequent CSF-1-stimulated [3H]TdR uptake as the cells reenter the cycle. Finally, we show that both human and mouse TNF-alpha increase CSF-1-stimulated log-phase growth and reentry of quiescent cells into the cycle equally on a molar basis (half-maximal stimulation of approximately 0.3 nM). The latter observation argues that the growth-stimulatory effects of TNF-alpha are mediated via the 55-60-kd TNF receptor. We conclude that TNF-alpha acts directly on growth-competent macrophages to decrease significantly the population doubling time in a manner that enhances the mitogenic effects of CSF-1.

Autocrine regulation of macrophage proliferation by tumor necrosis factor-alpha.

We have examined production of tumor necrosis factor-alpha (TNF-alpha) from mouse bone marrow-derived macrophages (BMM) after stimulation by lipopolysaccharide (LPS), macrophage colony-stimulating factor (M-CSF/CSF-1), or granulocyte-macrophage colony-stimulating factor (GM-CSF). To control for effects of trace levels of LPS in culture media, we examined CSF-1 and GM-CSF-stimulated TNF-alpha production in relatively homogeneous populations of normal LPS-hyporesponsive C3H/HeJ BMM and a growth factor-dependent cell line (S1) of C3H/HeJ origin. We found that both TNF-alpha mRNA and protein are induced in these macrophage populations by CSF-1 stimulation alone. Stimulation with GM-CSF, however, appears to negatively regulate TNF-alpha protein levels. Most TNF-alpha detectable after CSF-1 or GM-CSF stimulation was cell associated. Only stimulation by LPS resulted in large amounts of released TNF-alpha detectable in culture supernatant. The hypothesis that endogenously produced TNF-alpha can function as an autocrine growth factor for CSF-1-stimulated proliferation was supported by the demonstration of a partial inhibition of BMM proliferation (p < 0.05) in the presence of a neutralizing anti-TNF-alpha antiserum. These results demonstrate that CSF-1 alone is capable of inducing expression of both TNF-alpha mRNA and protein, that GM-CSF may negatively regulate TNF-alpha protein production, and that endogenously produced TNF-alpha may provide additional growth signals for CSF-1 mediated BMM proliferation.

Evaluation of reticuloendothelial function in autoimmune hemolytic anemia using an in vitro assay of monocyte-macrophage interaction with erythrocytes.

We have used an in vitro assay of monocyte-RBC interaction to study the correlation of in vitro monocyte activity with in vivo lysis in patients with autoimmune hemolytic anemia (AIHA). All of 16 patients with a positive direct antiglobulin test (DAT) (0.5 + to 4+) and clinical evidence of hemolysis showed elevated association (ARBC) and phagocytic (PRBC) indices. Of 6 patients studied with a positive DAT (0.5+ to 4+) without clinical evidence of hemolysis, none showed elevated PRBC while 2 showed slightly elevated ARBC. Thus, when using a PRBC index, our assay distinguished between hemolysing and non-hemolysing patients independent of the degree of red cell sensitization as determined by the DAT. In addition, we have studied 6 patients with a positive DAT following alpha-methyldopa therapy. Two of these patients were hemolysing, 4 were not. Again, our assay correlated with in vivo lysis. Finally, we have studied red cells from 11 patients with DAT-negative acquired hemolytic anemia. Seven of these patients showed elevated ARBC and PRBC indices, indicating a possible immune etiology involving extravascular lysis in some DAT-negative acquired hemolytic anemias.

HIV infection in vitro enhances the activity of src-family protein tyrosine kinases.

OBJECTIVE: We examined the effect of HIV infection on src-family protein tyrosine kinase (PTK) activity to determine if alterations in src-family PTK activity could contribute to the HIV-related chronic immune system activation observed in patients infected with HIV. METHODS: Jurkat, a CD4+ human T lymphocyte cell line was infected with HIV IIIB. Kinase activity was determined by in vitro immune complex kinase assays using antibodies specific for the src-family PTKs, p56lck, p59fyn and p60c-src expressed in T lymphocytes. PTK protein and total phosphotyrosine levels were assessed by Western blotting. The role of the gp120-CD4-Lck interaction in HIV-related PTK activation was determined using gp 120-treated Jurkat cells and HIV-infection of JCaM 1.6 cells, a Jurkat-derived cell line that lacks p56lck. RESULTS: Cells infected with HIV for 24 h exhibited increased levels of total tyrosine phosphorylation and enhanced src-family PTK activity without altered levels of expression of src-family kinases. The activity of Lck and Fyn was enhanced within 30 min of infection. HIV-related src-family PTK activation was not a function of the gp120-CD4-Lck interaction and occurred in the presence of 10 mmol/l zidovudine indicating that reverse transcriptase and activation of the HIV genome is not required. CONCLUSIONS: HIV-related activation of src-family PTK is a response of the cell to early stages of the virus life cycle, possibly either membrane fusion or viral uncoating. These results indicate that endogenous src-family PTKs may play a role in HIV-related immune activation and dysfunction. Moreover, activation of src-family PTK may be a mechanism used by the virus to facilitate some aspect of its own life cycle.

Differential expression of tumor necrosis factor-alpha isoforms from lipopolysaccharide- and cytokine-stimulated mouse macrophages.

Tumor necrosis factor-alpha (TNF alpha) is a biologically active cytokine with a wide range of functions, which is primarily expressed by macrophages. It is produced as a biologically active propeptide that becomes processed to the mature form of secreted protein. Previous studies used a mouse macrophage cell line and showed that after stimulation with lipopolysaccharide, TNF alpha propeptide is expressed as multiple isoforms with approximate molecular masses of 26, 29 and 32 kDa. However, little is known of the production of TNF alpha isoforms from normal macrophages or of the effects of cytokines on TNF alpha production by macrophages in the absence of co-stimulation by lipopolysaccharide. We have compared the TNF alpha isoforms produced by cytokine-and lipopolysaccharide-stimulated bone marrow-derived macrophages from mice that normally respond to lipopolysaccharide (C3H/HeN) and mice that are hyporesponsive (C3H/HeJ). We found that the pattern of immunoprecipitated TNF alpha propeptide isoforms expressed depended on the stimulus: lipopolysaccharide, granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor. Lipopolysaccharide induced three isoforms of 25, 29 and 35 kDa, supporting previous studies. However, macrophage and granulocyte-macrophage colony-stimulating factors also stimulated cells to express the 24 and 27 kDa isoforms, but not the 35 kDa isoform. In addition, cells stimulated with granulocyte-macrophage colony-stimulating factor expressed a novel 20 kDa propeptide. The results show that granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor and lipopolysaccharide differently regulate TNF alpha protein expression and suggest that different isoforms may have different functions.

A specific and reliable bioassay for the detection of femtomolar levels of human and murine tumor necrosis factors.

A reliable, highly sensitive, cytolytic bioassay for the quantitation of both human and murine tumor necrosis factor (TNF) is described. The assay is 2-180-fold more sensitive than other currently described bio- or immunoassays (limits of detection: 500 fg/ml (29 fmol/l) human TNF-alpha, 200 fg/ml (12 fmol/l) murine TNF-alpha and 130 fg/ml (7 fmol/l) human TNF-beta). The assay, which uses L929-8, a newly isolated subclone of the murine fibroblastoid cell line L929, detects human TNF-alpha approximately 180-fold more sensitively than previously described L929 subclone assays. Maximum sensitivity is obtained by preincubating L929-8 cells at 37 degrees C with 2 micrograms/ml actinomycin D (1-2 h), then culturing with TNF at 40 degrees C for 20 h in medium containing high serum (15% FBS). Relative viable cell content in 96-well microtiter plates is determined colorimetrically by uptake of the non-carcinogenic dye neutral red. Other cytokines have no effect, either alone or in combination with TNF. Cytokines tested were IL-1 through IL-6, GM-CSF, G-CSF, CSF-1, LIF, TGF-beta, NGF, Epo or IFN-gamma, LPS, PGE2, dexamethasone and cyclosporin A, also have no effect, either alone or in combination with TNF. L929-8 cells maintain the above sensitivity to TNF for at least 4 months in continuous culture. Thus, the assay allows rapid, inexpensive, reliable and specific quantitation of rodent and human TNFs. Its very high sensitivity should allow accurate detection of biologically active TNF in biological fluids such as human serum.

A new reagent (ZZAP) having multiple applications in immunohematology.

A reagent (ZZAP) containing a mixture of 0.1 M dithiothreitol (DTT) plus 0.1% cysteine-activated papain was found to dissociate IgG immunoglobulin from red blood cells (RBC) of patients having a positive direct antiglobulin test (DAT) although this could not be achieved with either chemical alone. In all 67 patients tested, ZZAP treatment of IgG sensitized RBC reduced the strength of the DAT, and in all 52 instances tested, this allowed for accurate Rh phenotyping using slide/rapid tube typing reagents. This included five examples in which spontaneous agglutination that occurred in saline or 6% albumin was eliminated by ZZAP. Thus, all red blood cell typing for Rh-Hr antigens was accomplished using slide/rapid tube reagents making unnecessary the use of saline reactive or chemically modified antisera. Kidd antigen typing is also possible after ZZAP treatment of IgG sensitized RBC. In regard to warm autoabsorption tests, ZZAP treatment of 14 RC samples having a positive DAT proved preferable to heat elution technics since equal or greater amounts of IgG were removed by ZZAP and little or no hemolysis resulted. ZZAP has no effect on ABH, Rh or Kidd antigens but denatures Duffy, MNSs and all Kell antigens tested (K:1-K:7, K:11-K:14, K:18, K:19). This should prove valuable in certain investigations of multiple alloantibodies and, moreover, may allow for better characterization of Kell antigens.

Erythrocyte age-fractionation using a Percoll-Renografin density gradient: application to autologous red cell antigen determinations in recently transfused patients.

A rapid technic for the age-fractionation of human erythrocytes into reticulocyte-enriched (young) red blood cells and reticulocyte-poor (old) red blood cells using an isopycnic density gradient centrifugation through Percoll-Renografin was evaluated for use in autologous red blood cell antigen determinations in multiply-transfused patients. The fractionation was demonstrated by statistically significant density-related changes in pyruvate kinase and acetylcholinesterase activities (P = 0.002 and 0.042, respectively) and by the distribution of reticulocytes on the gradient (P less than 0.005). With initial reticulocyte counts of less than or equal to 1.5%, reticulocyte counts up to 78% were achieved (means = 25%; n = 31). When starting with reticulocyte counts greater than 5%, samples containing up to 98% reticulocytes were obtained (means = 64%; n = 7). The technic requires less than two hours, uses isotonic media, and is nontoxic to red blood cells. Volumes of red blood cells up to 10 mL can be fractionated at one time and the gradient medium is stable when refrigerated at 4 degrees C. Red blood cell typing was performed in six patients who had received from 4-29 units of blood within a 12-hour period. Within 72 hours posttransfusion, typing of the reticulocyte enriched fraction correctly identified the patient's red blood cell antigens with all 16 antisera tested. This technic for typing reticulocyte-enriched samples is of importance for confirmation of antibody specificity in determining whether an antibody is an alloantibody or autoantibody, and in the selection of donor blood for transfusion to patients having autoimmune hemolytic anemia.

Globotriaosyl ceramide receptor function - where membrane structure and pathology intersect.

The glycosphingolipid globotriaosyl ceramide, (Galalpha1-4Galss1-4 glucosyl ceramide-Gb(3)) also known as CD77 and the P(k) blood group antigen, is bound by both verotoxins and by the HIV adhesin, gp120. Gb(3) plays an important receptor role in VT induced hemolytic uremic syndrome (HUS) and HIV infection. The organization of glycolipids, including Gb(3), into lipid rafts is central to both pathologies. The fatty acid heterogeneity within the Gb(3) lipid moiety plays a central role in assembly within such ordered domains. Differential binding of verotoxins and gp120 to such Gb(3) isoforms in model and cell membranes indicates a significant role in the eventual pathogenic outcome. HUS may provide the first example whereby membrane Gb(3) organization provides a predictor for tissue selective in vivo pathology.