Experimental allergic encephalomyelitis (EAE) has been used as an animal model for the human demyelinating disease multiple sclerosis (MS). In acute MS or EAE, early disruption in the integrity of the blood-brain-barrier (BBB) precedes brain infiltration by inflammatory cells or any clinical evidence of disease. BBB permeability could be affected by vasoactive mediators and cytokines released from perivascular brain mast cells. We investigated the number and degree of activation of brain mast cells in EAE and the effect of the heterocyclic histamine-1 receptor antagonist hydroxyzine, a piperazine compound known to also block mast cells. Acute EAE was induced in Lewis rats by immunization with whole guinea pig spinal cord homogenate and complete Freund's adjuvant (CFA). A second group of animals were treated orally with hydroxyzine for one day before immunization and then continuously for 14 days. Control rats were treated with CFA or hydroxyzine alone. The clinical progression of EAE was assessed on days 10, 12 and 14 after immunization. The number of metachromatic mast cells and the degree of degranulation was assessed in the thalamus with light microscopy. At day 14, there was a three-fold increase in the number of brain mast cells with EAE, as compared to controls. These cells were positive for the immunoglobulin E binding protein (FcepsilonRI), while those from control rats were not. Over 40% of all thalamic mast cells studied in EAE showed partial staining or extruded secretory granule indicative of secretion. Hydroxyzine treatment inhibited (p<0.05) the progression and severity of EAE by 50% and the extent of mast cell degranulation by 70% (p<0.05). These findings indicate that brain mast cells are associated with EAE development and that inhibition of their activation correlates positively with the clinical outcome.
The effect of thymulin on IL-2 receptor (IL-2R) expression by avian splenocytes was examined in the functionally hypothyroid sex-linked dwarf (SLD) and in normal euthyroid K strain chickens. Daily thymulin injections of 0, 0.05 and 5.0 ng/100 g body weight were given from hatching until 4 weeks of age. ConA-treated and non-stimulated splenocytes from these animals were analyzed by flow cytometry for their expression of IL-2Rα, CD4 and CD8 cell surface molecules. ConA activation increased the number of IL-2R+ cells within K strain more than in the SLD. Thymulin treatment increased the number of IL-2R+ cells in the SLD but had the opposite effect in K strain chickens. Mitogen activation or thymulin treatment had little effect on the IL-2R density within small cell populations. In contrast, mitogen activation increased the density of IL-2R on larger cell populations in both K and SLD. IL-2R densities on non-stimulated larger cells decreased in the SLD after thymulin exposure. Thymulin treatment produced no effect on the mean IL-2R densities for large activated cells. ConA stimulation increased the number of CD4+ cells in both strains. The density of CD4 expression was modulated by both mitogen activation and thymulin treatment. ConA stimulation produced an increase in the number of CD8+ cells. The SLD had fewer CD8+ cells than did the K strain and thymulin treatment had little effect on this population in either strain. Mitogen stimulation increased the density of CD8 on CD8+ cells but again thymulin treatment had little effect. These results suggest that thymulin can modulate IL-2R expression on splenocytes and that this effect may be dependent upon the thyroidal status of the animal. Further, these data suggest that thymulin has a differential effect on the CD4 and CD8 T-cell subpopulations.
It has been demonstrated that the synthetic immunostimulatory compound, adamantylamide dipeptide (AdDP) produces hematopoiesis-stimulating effects in mice exposed to sublethal doses of ionizing radiation and increases survival in experimental animals irradiated with a lethal dose. These findings might suggest contingent extension of clinical indications for the administration of AdDP for the conditions of hematopoietic suppression, especially in oncology.
Dialyzed leukocyte extract (DLE) (Immodin SEVAC, Czech Republic) was shown to enhance the recovery of the pools of hemopoietic stem cells (CFUs) and of granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) in the bone marrow in vivo, as well as to increase the numbers of leukocytes and thrombocytes in the peripheral blood of mice exposed to a sublethal dose of gamma-rays, with an ensuing increase in the numbers of mice surviving the lethal radiation dose. In experiments performed in vitro, DLE or sera of mice administered with DLE were added to cultures of intact mouse bone marrow cells containing suboptimal concentrations of hemopoietic stimulatory cytokines, namely recombinant mouse interleukin-3 (rmIL-3) or recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF); under these experimental conditions, both DLE and sera of mice administered DLE were found to increase the counts of GM-CFC colonies in the cultures. It can be hypothesized on the basis of the findings obtained in vitro that the described co-stimulating activity (CoSA) of DLE may play a role also under in vivo conditions; the enhancement of the recovery of hemopoiesis suppressed by ionizing radiation may be due to a co-operation of the stimulatory effects of DLE with the action of cytokines endogenously produced in irradiated tissues.
The present study was undertaken to investigate the effect of biphenyl dimethyl dicarboxylate (PMC) on the humoral immunosuppression by ethanol (EtOH) in ICR mice. PMC at a dose of 6 mg/kg was orally administered to mice daily for 28 consecutive days, and the control mice were given vehicle. Mice treated with EtOH were given freely with 20% EtOH instead of water. The results of this study are summarized as follows; a gain of body weight and the relative weights of spleen and liver were significantly increased by combination of PMC and EtOH, as compared with those in mice treated with EtOH alone. Splenic plaque forming cells (PFC) and hemagglutination (HA) titers to sheep red blood cells (SRBC), and the secondary IgG antibody response to bovine serum albumin (BSA) were decreased by the treatment of EtOH alone, then restored to normal level by PMC treatment. The elevations of serum glutamic-pyruvic transaminase (S-GPT) and total protein levels caused by EtOH were reduced to normal level by the combination of PMC and EtOH. In addition, lower serum albumin and A/G ratio were also increased to normal level. These findings indicate that PMC has a protective effect against EtOH-induced humoral immunosuppression.
It is well established that glucocorticoid hormones induce apoptosis in immature developing thymocytes. Thymocyte apoptotsis can be modulated by growth factors, anti-oxidants and adhesion receptors. We have previously demonstrated that thymosin α1 (Tα1) antagonizes dexamethasone-induced apoptosis of CD4+CD8+ thymocytes. In the present study, we further characterize the dose and time dependence of Tα1's antagonism of dexamethasone-induced thymocyte apoptosis. Tα1 is effective at concentrations ranging from 2 to 100 μg/106 thymocytes. Tα1 pre-treatment is necessary to achieve its anti-apoptotic activity. Tα1 provides temporary protection to thymocytes by slowing dexamethasone's apoptotic activity up to 12 h post dexamethasone treatment. Additionally, Tα1's activity is not sensitive to cycloheximide treatment, suggesting Tα1's activity is independent of protein synthesis. Finally, Tα1 is unable to antagonize apoptosis induced by the reactive oxygen species, H2O2, suggesting Tα1's antagonism of dexamethasone occurs at the early stages of dexamethasone-induced apoptosis, prior to the production of reactive oxygen species. This evidence suggests that Tα1 may provide a mechanism to transiently extend the life of a thymocyte during thymic selection.
It has been previously reported that endogenous insulin levels decrease during tumor growth. We have now studied whether this host endocrine response is independent of the way in which the tumor is induced. For this purpose, animals transplanted with tumor cells induced by 3-methylcholanthrene (MCA) or 7,12-dimethylbenz(a) anthracene (DMBA), or with EL-4 lymphoma cells, and animals that develop autochthonus tumors induced by MCA or the murine mammary tumor virus (MMTV) were used. These procedures result on the induction of tumors of different histologic types: fibrosarcoma, mammary adenocarcinoma and lymphoma. The results obtained showed that a reduction in insulin levels preceded the overt appearance of tumors in all models of syngeneic or autochthonus tumors studied but not when DMBA-induced tumor cells were administered into allogeneic recipients. Reduced levels of insulin before tumor detection appeared to affect the onset of MCA-induced tumors. Indeed, those mice with a late tumor onset were those that had a more pronounced decrease in insulin blood levels during the induction phase of authocthonous MCA-induced tumors. Soluble factors associated with tumor growth seem to mediate the reduction in insulin blood levels in mice transplanted with EL-4 tumor cells. The results obtained indicate that the reduction in insulin levels detected is a consequence of the recognition of tumor cells by the host, and seems to be independent of the histologic type of the neoplastic cells that develop. Pharmacological interventions at the levels of mechanisms that control insulin output should clarify the relevance of decreased levels of this hormone for tumor development.
Polymyxin B (PMB), an antibiotic with anti-endotoxin activity, was used to examine the participation of endogenously produced endotoxin in the enhancement of recombinant human tumor necrosis factor (rhTNF)-induced toxicity in d-galactosamine (GalN)-sensitized mice. GalN-sensitized mice (700 mg/kg, intraperitoneally (i.p.)) injected together with rhTNF (1×104 U/mouse, intravenously (i.v.)) exhibited severe symptoms, with 100% mortality at 18 h. However, mice pretreated with PMB (20 mg/kg, i.p.) showed protection against the rhTNF-induced lethality following GalN sensitization. Little or no effects were observed on alanine aminotransferase (ALT) activity or lactate dehydrogenase (LDH) isozyme leakage in serum in mice 7 h after administration of rhTNF alone. Administration of rhTNF to GalN-sensitized mice resulted in marked increases in ALT activity and LDH isozyme leakage relative to those in mice treated with rhTNF alone. In mice pretreated with PMB, the levels of ALT and LDH isozyme leakage 7 h after rhTNF/GalN injection were significant decreased as compared with those in mice treated with rhTNF/GalN. Similarly, injection of PMB markedly decreased lipid peroxide formation in the liver of the GalN-sensitized mice treated with rhTNF. The injection of a low endotoxin dose (0.1 mg/kg, i.p.) markedly increased the lethality in mice treated with rhTNF (5×103 U/mouse, i.v.) and GalN, and these animals showed 100% mortality at 8 h. These findings suggested that the extent of TNF-induced toxicity caused by GalN administration may be a result of synergism between TNF and gut-derived endotoxin. It is likely that endogenously produced endotoxin play a significant role in rhTNF/GalN-hypersensitized mice.
It has been demonstrated that tyrosine kinase (TK) and phosphatidylinositol 3-kinase (PI3-K) are involved in IgE-mediated stimulation of human basophils; conversely, little is known about the biochemical pathways activated by IL-3 and GM-CSF. The aim of this study was to evaluate the effects of TK and PI3-K inhibitors on basophil histamine release induced by anti-IgE, IL-3 and GM-CSF. Since IL-3 and GM-CSF cause histamine release from normal human basophils only when the inhibitory effect of extracellular Na(+) has been removed, peripheral blood leukocytes were suspended in isotonic solutions containing either 140 mM NaCl or 140 mM N-methyl-D-glucamine(+). After stimulation with anti-IgE, IL-3 or GM-CSF, histamine release was measured by an automated fluorometric method. The effects of preincubation with four different TK inhibitors (AG-126, genistein, lavendustin A, tyrphostin 51) and one PI3-K inhibitor (wortmannin) were evaluated. AG-126, genistein and lavendustin A exerted a significant dose-dependent inhibitory effect on basophil histamine release induced by anti-IgE (either in high or in low Na(+) medium), IL-3 and GM-CSF. Among the TK inhibitors, lavendustin A exerted the most potent activity, followed by AG-126 and genistein. Tyrphostin 51 caused a weak inhibition of histamine release induced by IL-3, GM-CSF and anti-IgE in a low Na(+) medium, but not in a physiological Na(+)-containing medium. The PI3-K inhibitor wortmannin exerted the most effective inhibitory activity on the histamine release induced by the three agonists. The combined effects of lavendustin A and wortmannin were less than additive, suggesting that TK and PI3-K are involved in the same activation pathway in human basophils. These results suggest a possible role of TK and PI3-K in basophil histamine release induced by anti-IgE, IL-3 and GM-CSF. TK and PI3-K are indeed potential therapeutic targets for antiallergic drugs.
The transcription factor NF-kappaB is the central regulator for the expression of various genes involved in inflammation, infection and immune response including the genes for IL-1beta, TNF-alpha, IL-6 and leukocyte adhesion molecules. Here, we show that the anti-allergic drug histaglobin down-regulates the release of IL-1beta, TNF-alpha, IL-6 and IL-10 in human peripheral blood mononuclear cell cultures. This down-regulatory effect becomes even more pronounced when the cultures are simultaneously activated with the T-lymphocyte mitogen phytohemagglutinin (PHA) or with the B-lymphocyte and macrophage activator lipopeptide (P(3)CSK(4)). We also demonstrate that histaglobin inhibits the nuclear translocation of NF-kappaB in response to TNF-alpha or lipopolysaccharide (LPS) in bone marrow-derived macrophages of Balb/c mice. The inhibitory effect of histaglobin on NF-kappaB activation and cytokine release might be responsible for its anti-allergic effect as demonstrated in clinical studies.
Multiple immune system actions have been ascribed to paclitaxel (taxol), a novel anticancer drug, including the capacity to induce macrophage antitumor cytotoxic molecule production. In the present studies, we demonstrated that paclitaxel produced a selective inhibition of lipopolysaccharide (LPS)-induced B cell proliferation. Similarly, in vitro polyclonal antibody-forming cell responses also were found to be inhibited by paclitaxel. Conversely, paclitaxel exhibited no inhibitory effects on concanavalin A (Con A)-induced T cell proliferation. To study the pathway leading to paclitaxel-induced immunosuppression, we analyzed Raf-1/ERK and JNK/p38 MAPK pathways, both of which have been reported to be involved in LPS signaling. Our results indicate that taxol treatment inhibits Raf-1 kinase activation while having no effect on ERK activation suggesting that ERK activation is distinct from upstream Raf-1 kinase in taxol-induced immunomodulation. Furthermore, paclitaxel pretreatment caused down-regulation of stress-activated MAPKs, JNK and p38 MAPK in lipopolysaccharide (LPS)-stimulated mouse splenic lymphocytes, demonstrating that spleen cells are induced to a state hyporesponsive to LPS stimulation by pre-exposing them to paclitaxel. Taken together, these results suggest that down-regulation of JNK/p38 MAP kinase may contribute to paclitaxel-induced immunosuppression in mouse splenic lymphocytes.
We determined whether the expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP) in murine macrophages is regulated by the novel synthetic bacterial lipopeptide JBT 3002. Multilamellar liposomes (MLV) encapsulating JBT 3002 (MLV-JBT 3002) stimulated the production of 72-kDa and 92-kDa (gelatinase A and B) type IV collagenase and inhibited the production of murine metalloelastase (MME) in a dose-dependent manner in murine peritoneal macrophages. MLV-JBT 3002 also induced production of TIMP-1. MLV-JBT 3002 did not induce collagenase production in tumor cells. Priming murine macrophages with interferon-gamma (IFN-γ) inhibited JBT 3002-stimulated production of both MMP-9 and MMP-2 and further inhibited production of MME by a mechanism involving nitric oxide (NO). This conclusion is based on data showing that IFN-γ failed to inhibit production of MMP in the presence of l-methyl arginine or in macrophages from inducible nitric oxide synthase knockout mice. These data suggest that JBT 3002 differentially regulates the production of various MMPs and TIMP in macrophages.
Basic secretagogues, such as compound 48/80, stimulate secretion in rat peritoneal mast cells by directly activating the heterotrimeric G-protein Gi3 (Aridor M, et al. Science 1993;262:1569–72). Cultured RBL-2H3 mast cells do not normally respond to basic secretagogues, but acquire such responsiveness upon prolonged exposure to the kinase inhibitor, quercetin, which also increases the cellular level of Gi3 (Senyshyn J, Baumgartner RA, Beaven MA. J Immunol 1998;160:5136–44). Expression of a GTPase-deficient mutant of Gαi3 in RBL-2H3 cells results in the stimulation of Ca2+-triggered exocytosis and release of arachidonic acid (AA) (Zussman A, Hermuet S, Sagi-Eisenberg R. Eur J Biochem 1998;258:144–6). Here we show that long-term incubation with quercetin markedly stimulates Ca2+-triggered exocytosis and release of AA from the RBL-2H3 cells. We further show that membranes derived from such quercetin-treated cells display a reduced GTPase, but not ATPase, activity. Taken together with our previous observations, these results further implicate Gi3 as one of the cellular targets through which quercetin confers responsiveness towards the family of basic secretagogues.
Nitric oxide (NO) plays various important roles in the physiological system. With regard to chemotaxis of neutrophils, there are reports that endogenous NO is a mediator of chemotaxis, and others that exogenous NO inhibits chemotaxis. It is also reported that NO itself expressed chemotactic activity. On the other hand, we have recently proposed the importance of cofilin, an actin-binding phosphoprotein, in phagocyte functions through dephosphorylation and translocation to the plasma membrane regions. Because chemotaxis is a phenomenon of dynamic cell movement, cofilin, a regulator of the cytoskeletal system, may be involved in its mechanisms. To clarify further the effect of NO on functions of leukocytes and to examine the effect of NO on cofilin, we investigated the chemotaxis of neutrophil-like HL-60 cells induced by NO, as well as the influence of NO on the phosphorylation and intracellular distribution of cofilin. Two NO donors, 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamine (NOC5) and S-nitroso-N-acetylpenicillamine (SNAP), were shown to cause chemotaxis, and, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO), a NO-specific scavenger, inhibited the chemotaxis induced by NO-donors, suggesting that NO itself released from the NO donors has chemotactic activity. LY-83583 and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), inhibitors of soluble guanylate cyclase, inhibited the chemotaxis to NO donors, which implies that soluble guanylate cyclase is involved in the signaling pathway of this NO action. We also found that NO caused translocation of cofilin to the cell periphery, though dephosphorylation of cofilin was not detected. These results demonstrate that NO has chemotactic activity for neutrophils and caused the translocation of cofilin to the plasma membrane regions without its dephosphorylation.
To analyze the immunomodulatory effect of pyrrolidine dithiocarbamate (PDTC) on the endotoxin (LPS) stimulated inflammatory response, we measured the LPS-stimulated cytokine and NO production in murine peritoneal macrophages, J774A.1 cells and human whole blood in the presence of PDTC (60 microM). PDTC significantly inhibited the production of nitrite, IL-1beta and IL-6 in these cells. TNFalpha release was stimulated in murine cells, but suppressed in human whole blood. We further investigated the influence of PDTC on mortality and cytokine release in mouse endotoxin shock. PDTC was i.p. injected 30 min prior to the induction of endotoxin shock in female NMRI-mice and survival was significantly improved as compared to controls (48% vs 20%, n=25 per group). Plasma concentrations of TNFalpha were slightly augmented while IL-6 levels were decreased in PDTC-treated animals as compared to controls, however, without reaching significance. We conclude that PDTC is a potent immunomodulatory substance that modulates the inflammatory response in vitro and reduces mortality in mouse endotoxin shock. The pathophysiological mechanisms of the protective effect of PDTC in vivo, however, appears to be pluripotent, comprising both antioxidative properties and the inhibition of NF-kB.
Synthetic lipopeptides derived from the N-terminus of bacterial lipoprotein constitute potent macrophage activators and polyclonal B-lymphocyte stimulators. They are also efficient immunoadjuvants in parenteral, oral and nasal immunization either in combination with or after covalent linkage to an antigen. Here we show how alterations in the molecular structure influence their biological properties indicating P3CSK4 as one of the most active members of a lipopentapeptide fatty acid library. This compound resulted in a most pronounced macrophage stimulation as indicated by NO release, activation of NFkappaB translocation, and enhancement of tyrosine protein phosphorylation. Furthermore, P3CSK4 activates/represses an array of at least 140 genes partly involved in signal transduction and regulation of the immune response. Finally we have evidence that P3CSK4 constitutes an effective adjuvant for DNA immunizations, especially increasing weak humoral immune responses. Our findings are of importance for further optimizing both conventional and genetic immunization, and for the development of novel synthetic vaccines.
This study was undertaken to evaluate the effect of recombinant Schistosoma mansoni-26 Glutathione S-transferase (rSm 26 GST) or soluble egg antigen (SEA) alone and in addition to praziquantel (PZQ) on the state of resistance to S. mansoni reinfection. The associated changes in the immune responses were evaluated. The experimental group of mice were injected intravenously before S. mansoni infection (80 cercariae/mouse) either with rSm26 GST (1 microgx4) or SEA (10 microgx4) in addition to PZQ (2x500 mg/kg) administered 6 weeks post-infection. Seven control groups were used, three of them were the infected (80 cercariae/mouse), the challenged (240 cercariae/mouse) and the infected challenged controls (80+240 cercariae/mouse). The rest of the four groups were the treated controls receiving: the GST-Lyzate, rSmGST, SEA and PZQ in the same doses and at the same timings. Challenge infection was conducted for all the groups 8 weeks post-infection. Animals were sacrificed 3 weeks post-challenge. After sacrifice animals were perfused and percentage resistance to reinfection was calculated. Immune responses were assessed by the measurement of hepatic granuloma diameter, intralesional T-cell phenotypes and serum immunoglobulin isotypes. The highest percentage of resistance to reinfection was observed in rGST-treated group while the lowest percentage of resistance was detected in PZQ-treated group. Whereas in mice receiving combined rGST or SEA and PZQ, percentage resistance to reinfection was significantly higher than that in PZQ treated mice. The remarkable reduction in granuloma diameter in rGST-treated group with or without PZQ was associated with decrease in the intralesional L(3)T(4)(+) and increase in Lyt(2)(+) T-cell phenotypes. However, no special relationship was observed between the percentage of resistance and the changes in granuloma diameter or intralesional T-cell phenotypes. The increase in percentage resistance to reinfection was found accompanied by increased anti SWAP IgE. Combined rGST and PZQ provided the complementary goals of improved state of resistance to reinfection 'which was compromized after cure with PZQ' and the maximal reduction in granuloma diameter.
L-deprenyl, a monoamine oxidase-B inhibitor, partially reversed the age-associated decline in splenic sympathetic noradrenergic (NA) innervation and immune reactivity in old male rats. The purpose of the present study was to examine whether the effects of deprenyl on splenic sympathetic NA nerve fibers and immune functions are mediated through a metabolite of deprenyl, L-desmethyldeprenyl. Old male F344 rats were treated with 0, 0.25, or 1.0 mg L-(-)-deprenyl/kg BW; 0.025, 0.25, or 1.0 mg L-(-)-desmethyldeprenyl/kg BW; and 1.0 mg D-(+)-desmethyldeprenyl/kg BW i.p. daily for 8 weeks. The animals were sacrificed after a 10-day drug wash-out period and the spleens were removed for histofluorescence, immunocytochemistry, neurochemical, and immunological analysis. The volume density of NA nerve fibers was increased in the spleens of deprenyl- and L-desmethyldeprenyl-treated old rats. Con A-induced IFN-gamma production by spleen cells was elevated in 1.0 mg/kg deprenyl- and L-desmethyldeprenyl-treated rats in comparison to saline- and D-desmethyldeprenyl-treated old rats. Deprenyl and desmethyldeprenyl treatment did not alter the percentage of CD5+ T cells, but treatment with 1.0 mg/kg deprenyl and 0.025 mg/kg L-desmethyldeprenyl prevented the decline in the percentage of sIgM(+)B cells in the spleens of old rats. These results suggest that L-desmethyldeprenyl may be as equipotent as deprenyl in preventing age-associated diminution in splenic sympathetic NA innervation and immunocompetence.
