PI3K/mTOR Inhibitor Dactolisib Attenuates Allergic Response Through Inhibitions of the Sensitization and Mast Cell Activation.

The aim of this study was to evaluate the anti-allergic potentials of dactolisib, a dual PI3K/mTOR kinase inhibitor, on two important events for allergy: sensitization and the onset of anaphylactic symptoms. After sensitization with the antigen ovalbumin (OVA), five successive oral administrations of dactolisib effectively decreased serum anti-OVA antibody-an indicator of sensitization-levels in mice. In parallel with the antibody levels in their serum, anaphylactic rectal temperature decrease induced by the re-administration of OVA to dactolisib-treated mice was strongly diminished compared to that in vehicle-treated mice. The inhibitor also inhibited ex vivo splenic B cell activation indicated by the increase of phosphorylation of Akt, CD69 expression levels, and proliferation upon anti-B cell receptor antibody treatment, suggesting that suppressive effects of the inhibitor on B cell activation plays a role in its ability to decrease sensitization in vivo. We concurrently observed the anti-anaphylactic ability of dactolisib in vivoand in vitro. A single oral administration of the inhibitor attenuated the anaphylactic rectal temperature decrease induced in a mouse model of passive systemic anaphylaxis. In in vitro mast cell models, pretreatment with the drug inhibited the degranulation response and cytokine production in RBL2H3 cells triggered by IgE and antigens, without affecting cell viability. These results suggest that dactolisib, as well as other PI3K/mTOR inhibitors, might be a good candidate for anti-allergic drugs that exhibit both anti-sensitizing and anti-anaphylactic effects.

Anti-allergic effect of the Src family kinase inhibitor saracatinib.

The aim of this study was to evaluate the anti-anaphylactic and anti-allergic potentials of saracatinib, a Src family kinase inhibitor that was already shown to be safe in clinical trials when it was used as an anti-cancer drug. Using in vitro mast cell models, we found that saracatinib inhibited the degranulation response and cytokine production in RBL2H3 cells that were stimulated with IgE and antigen without affecting cell viability. Phosphorylation of Lyn, Akt, a PI3K substrate, and MAPKs including ERK, JNK, and p38, as well as the intracellular Ca2+ increase induced by this stimulation were also suppressed by saracatinib. This drug also inhibited symptoms in our established anaphylaxis mouse model, anaphylaxis-dependent spotted distribution of immune complex in skin (ASDIS). The intravenous injection of the mixture of IgE and antigen induced acute spotted distribution of immune complex in skin in hairless HR-1 mice, and its inhibition by intradermal injection of saracatinib was observed. Moreover, toluidine blue-stained skin sections indicated that the degranulation ratio of dermal mast cells was reduced in saracatinib-treated skin compared with vehicle-treated skin. Because only a few signaling inhibitors are used as anti-anaphylaxis and anti-allergic drugs, these results indicated the valuable suggestion that saracatinib and the Src family kinase inhibitors are good candidates for anti-anaphylaxis and anti-allergic drugs.

A rare case of dual origin of the left vertebral artery without convergence.

A case of dual origin of the left vertebral artery was encountered in a dissection course for medical students in 2014. Two vertebral arteries were observed on the left side. One arose from the aortic arch between the origin of the left common carotid artery and the left subclavian artery, entered the transverse foramen of the 4th cervical vertebra, and coursed upward into the transverse foramen. The other arose from the left subclavian artery as expected, divided into two branches anterior to the cervical vertebrae, and entered the transverse foramina of the 6th and 7th cervical vertebrae. Both branches flowed into the anterior spinal artery. Moreover, as seen in other anomalies, 3 arterial fenestrations were observed in the cranial arteries. This case is extremely unique with respect to the following points: the 2 ipsilateral vertebral arteries did not combine to form 1 vertebral artery, the vertebral artery of subclavian artery origin entered the transverse foramen of the 7th cervical vertebra, and 3 fenestrations were observed in the intracranial arteries. This is a very suggestive case for neurosurgeons and radiologists who perform treatments involving the vertebral artery.

Staff attitudes towards people with intellectual disabilities in Japan and the United States.

BACKGROUND: Staff attitudes may affect choices available to persons with intellectual disabilities (ID). This study examined attitudes towards people with ID among staff working with people with ID in Japan and the United States. METHOD: Attitudes of staff working with people with ID in Japan and the United States were compared using the Community Living Attitudes Scale, Intellectual Disabilities Form. Responses were examined via multivariate analysis of variance. RESULTS: In unadjusted analyses, Japanese staff exhibited a greater tendency towards Sheltering and Exclusion of people with ID and lower endorsement of Empowerment and Similarity of people with ID. After controlling for covariates, the country effect was no longer significant for Sheltering and Exclusion. Age and education were significantly associated with attitudes in the adjusted model. CONCLUSIONS: While attitudes in Japan appeared less supportive of community inclusion of people with ID, some of the differences between countries were attributable to other staff characteristics such as age and education. Findings provide new information about how attitudes of staff in each country compare with each other.

Tyrosine kinase inhibitor sunitinib relieves systemic and oral antigen-induced anaphylaxes in mice.

BACKGROUND: Systemic and oral antigen-induced anaphylaxes are mediated by immunoglobulin (Ig) E and mast cells, but there is no satisfactory treatment for the life-threatening allergic reaction. We investigated the potential of the multitargeted receptor tyrosine kinase inhibitor sunitinib to relieve anaphylactic reactions in food allergy and systemic anaphylaxis. METHODS: Efficacy of oral sunitinib on oral and parenteral antigen-induced anaphylaxes in Balb/c mice was evaluated. IgE-dependent degranulation and growth of rat basophilic leukemia RBL2H3 and bone marrow-derived mast cells (BMMCs) in response to sunitinib were investigated. RESULTS: Daily administration of sunitinib throughout antigen challenges prevented oral antigen-induced anaphylaxis including diarrhea, anaphylactic symptoms, and hypothermia. The mouse mast cell protease (MMCP)-1 concentration in serum and mast cell number in intestinal tissue after challenge were also decreased by the treatment. Spleen cells from sunitinib-treated mice contained smaller numbers of antigen-specific IgG-producing cells and secreted lower amounts of both Th1 and Th2 cytokines than those of the control mice, whereas the levels of antigen-specific antibodies in serum were not decreased. The reactions and MMCP-1 release in oral antigen-induced anaphylaxis and passive systemic anaphylaxis were attenuated even by a single predose of sunitinib. Degranulation and growth of RBL2H3 cells and BMMCs were greatly reduced by sunitinib. CONCLUSION: These results suggested that sunitinib relieves systemic and oral antigen-induced anaphylaxes by the prevention of mast cell activation and hyperplasia in intestinal tissue directly and indirectly through an immunosuppressive effect. Sunitinib and its related kinase inhibitors might be potential drugs for the treatment of food allergy and systemic anaphylaxis.

Preventive and therapeutic effects of rapamycin, a mammalian target of rapamycin inhibitor, on food allergy in mice.

BACKGROUND: Because few curative treatments are available for food allergy, we investigated the therapeutic potential of rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, on mouse food allergy. METHODS: The preventive and therapeutic effects of oral rapamycin on anaphylactic symptoms induced by oral ovalbumin (OVA) challenge in food allergy mice were investigated. Mast cell functions in response to rapamycin were also measured in the passive systemic anaphylaxis model and bone marrow-derived mast cells (BMMCs). RESULTS: Daily rapamycin from the first challenge (preventive protocol) attenuated food allergy symptoms including diarrhea, anaphylactic reactions, and hypothermia in mice. The treatment decreased the challenge-induced increases in mouse mast cell protease-1 in serum and mast cell numbers in the intestine. Notably, the mice that already showed food allergy symptoms by previous challenges recovered from the disease with daily administration of rapamycin (therapeutic protocol). Anti-OVA IgG1 and IgE levels in serum, as well as IFN-γ, IL-4, IL-13, IL-9, IL-10, and IL-17 secretion from splenocytes, were decreased by the treatments. In contrast, a single dose of rapamycin failed to affect passive systemic anaphylaxis. Spontaneous and IL-9-dependent survival and IgE-induced IL-13 secretion, but not degranulation, of BMMCs were reduced by rapamycin. CONCLUSION: Our data show that mouse food allergy was attenuated by rapamycin through an immunosuppressive effect and inhibition of intestinal mast cell hyperplasia. Inhibition of the IL-9 production-mast cell survival axis is one of the mechanisms of the therapeutic effect of rapamycin. Rapamycin and other mTOR inhibitors might be good candidates for therapeutic drugs for food allergy.

Aspergillus oryzae lectin induces anaphylactoid oedema and mast cell activation through its interaction with fucose of mast cell-bound non-specific IgE.

We investigated whether Aspergillus oryzae lectin (AOL), a fucose-specific lectin, induces anaphylactoid reactions and mast cell activation. The injection of AOL into footpads of mice produced a dose-related acute paw oedema. The AOL-induced oedema was attenuated by predose of histamine H1 receptor blocker or pretreatment of the lectin with fucose before injection and was not observed in SCID and mast cell-deficient WBB6F1-W/Wv mice. These results suggested that the AOL-induced anaphylactoid reaction was mediated by histamine released from mast cells. In addition, the activation of mast cells was seemed to be induced by the crosslinking of IgE on the cell surface following the binding of AOL to fucose residues in IgE. Consistent with the in vivo results, AOL induced the degranulation of the rat mast cell line RBL2H3 sensitized with monoclonal IgE. As AOL induced the increase in intracellular Ca(2+) concentration of IgE-sensitized RBL2H3 cells as well as antigen stimulation, AOL could input signals from FcεRI. The degranulation of IgE-sensitized RBL2H3 cells by AOL was diminished by pretreatment of AOL with fucose. Defucosylated IgE did not induce degranulation of RBL2H3 cells in response to AOL stimulation, in spite of its ability to induce degranulation by antigen stimulation as intact IgE. These results indicated that AOL bound to fucose residue of IgE causing antigen-independent IgE-mediated mast cell activation and anaphylactoid reactions in vitro and in vivo, respectively. AOL bound to human IgE as well as to mouse IgE, suggesting the possible implication of AOL in the allergic response to Aspergillus oryzae in humans.

Obesity and obesity-related secondary conditions in adolescents with intellectual/developmental disabilities.

BACKGROUND: To explore the prevalence of obesity and related secondary conditions associated with obesity in adolescents with intellectual/developmental disabilities (IDD). METHODS: In total, 461 parents of adolescents with IDD (M = 14.9 year, SD = 1.9) across 49 US states completed a web-based survey containing questions related to their child's health status, including body weight and existing health conditions. Results were compared with published data for youth without disabilities. RESULTS: Adolescents with autism and Down syndrome were two to three times more likely to be obese than adolescents in the general population. Secondary health conditions were higher in obese adolescents with IDD compared with healthy weight adolescents with IDD including high blood pressure, high blood cholesterol, diabetes, depression, fatigue, liver or gallbladder problems, low self-esteem, preoccupation with weight, early maturation and pressure sores. CONCLUSION: Obesity is as much of a health problem in youth with IDD as it is among youth without disabilities and, in certain disability groups, is a significantly greater health problem. Obese youth with IDD have a high number of obesity-related secondary conditions predisposing them to greater health problems as they transition into adulthood. Federal and local initiatives to reduce obesity among youth in the general population must recognise the need for interventions that are also relevant (i.e. accessible and effective) for youth with IDD.

Antioxidative role of interleukin-6 in septic lung injury in mice.

We have previously demonstrated the protective role of interleukin (IL)-6 against septic lung injury induced by lipopolysaccharide (LPS) using IL-6 knock-out (-/-) mice. This protection is mediated, at least partly, through the inhibition of the enhanced local expression of proinflammatory cytokines. In the present study, we addressed whether IL-6 regulates oxidative stress in the lung generated by LPS exposure using IL-6 (-/-) and corresponding wild type (WT) mice. Intraperitoneal LPS (1 mg/kg) challenge induced transcriptional expressions of inducible nitric oxide synthase and heme oxygenase -1 in the lung of mice with both genotypes. In the presence of LPS, these expressions were significantly greater in IL-6 (-/-) than in WT mice. Immunohistochemistry also showed that LPS induced a significant increase in 8-hydroxy-2'-deoxyguanosine formation in the lung as compared to vehicle. Furthermore, the formation was more intense in IL-6 (-/-) than in WT mice in the presence of LPS challenge. In the presence of LPS, lipid peroxidation in the lung was significantly greater in IL-6 (-/-) than in WT mice. These data suggest that the possible mechanisms in which endogenous IL-6 protects against septic lung injury induced by LPS involve, at least in part, its antioxidative properties.

Pulmonary exposure to diesel exhaust particle components enhances circulatory chemokines during lung inflammation.

This study examines the effects of DEP components on circulatory CC and CXC chemokines, potent activators and chemoattractants for macrophage and leukocyte subpopulations, in a murine model of lung inflammation. ICR mice were divided into six experimental groups which received intratracheal inoculation of vehicle, LPS alone (2.5 mg/kg), organic chemicals in DEP (DEP-OC: 4 mg/kg) extracted with dichloromethane, residual carbonaceous nuclei after the extraction (washed DEP: 4 mg/kg), DEP-OC + LPS, or washed DEP + LPS. Intratracheal instillation of each DEP component alone did not significantly change the circulatory level of macrophage inflammatory protein (MIP)-1alpha, MIP-2, and macrophage chemoattractant protein-1 (MCP-1) 24 h after the exposure as compared with vehicle instilled alone. In the LPS group, MCP-1, but not MIP-1alpha or MIP-2, was significantly greater than in the vehicle group. The combined administration of LPS and washed DEP caused a further three to five-fold increase in MIP-1alpha, MIP-2, and MCP-1 proteins in the serum as compared with LPS administered alone. No significant difference between the LPS + DEP-OC group and the LPS group was observed. These results indicate that pulmonary exposure to washed DEP enhances circulatory level of chemokines during lung inflammation. The enhancement may be important in the aggravations of systemic inflammatory responses and ischemic cardiovascular conditions associated with air pollution.

Effects of nanoparticles on lung physiology in the presence or absence of antigen.

Ambient particulate matter (PM) exacerbates allergic airway diseases. Our previous study showed that diesel exhaust particles, the main constituents in urban PM, enhance airway hyperresponsivness in mice. In addition, health effects of PM with a diameter of less than 100 nm, called nanoparticles, have been reported, and we have also demonstrated that carbon nanoparticles exacerbate antigen-related airway inflammation. The present study investigates the effects of pulmonary exposure to two sizes of carbon nanoparticles on lung physiology and lung expression of Muc5ac in the presence or absence of antigen in mice. Nanoparticles alone or ovalbumin (OVA) alone moderately enhanced cholinergic airway reactivity, as assessed by total respiratory system resistance (R) and Newtonian resistance (Rn). In the nanoparticle + OVA groups, all the parameters for lung responsiveness, such as R, compliance, elastance, Rn, tissue damping, and tissue elastance, were worse than those in the vehicle group, the corresponding nanoparticle groups or the OVA group. The lung mRNA level for Muc5ac was significantly higher in the OVA group than in the vehicle group, and further increased in the nanoparticle + OVA groups than in the OVA or the nanoparticle groups. These data suggest that carbon nanoparticles can enhance lung hyperresponsiveness, especially in the presence of antigen. The effects may be mediated, at least partly, through the enhanced lung expression of Muc5ac.

Heterogeneities in the biological and biochemical functions of Smad2 and Smad4 mutants naturally occurring in human lung cancers.

Smad family members are essential intracellular signaling components of the transforming growth factor-beta (TGF-beta) superfamily involved in a range of biological activities. The loss of sensitivity to TGF-beta is frequent in human lung cancers and inactivation of Smad family members are thought to play important roles in disruption of TGF-beta signaling. In the study presented here, we characterized the biological and biochemical functions of six Smad2 and Smad4 mutants, which we previously identified in human lung cancers. All mutant Smad2 and Smad4 were in fact found to be defective in transmitting growth inhibitory signals originating from TGF-beta and incapable of activating Smad/hFAST-1-mediated transcription. Transcriptional activation of plasminogen activator inhibitor type 1 (PAI-1) was impaired in four of the six mutants due to the defects in homo- and/or hetero-oligomerization with wild-type Smads. In contrast, the remaining two Smad mutants showed a modest reduction in the PAI-1 transcriptional activation and apparently retained the ability to oligomerize with wild-type Smads. Significant loss of growth inhibition and Smad/hFAST-1-mediated transcriptional activation by all of the six mutants suggested that Smad mutants are indeed functionally impaired Smad mutations and may play a role in lung tumorigenesis. Moreover, the present findings suggest that in addition to the impairment in the homo- and/or hetero-oligomerization, there may be an alternative mechanism producing disruption of TGF-beta signaling, involving hFAST-1-or possibly other transcriptional cofactor(s)-mediated transcriptional activation.

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells.

We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.

Effect of varying types of anti-arthritic drugs on Th1 and Th2 immune responses in mice.

The present study was undertaken to study the effect of varying types of anti-arthritic drugs on Th1 and Th2 immune responses in mice. To immunize mice, ovalbumin (OVA) emulsified with complete Freund's adjuvant was injected s.c. at the base of the tail (day 0). Indomethacin (IND) as a non-steroidal antiinflammatory drug (NSAID), dexamethasone (DEX) as a steroidal antiinflammatory drug, methotrexate (MTX), auranofin (AUR), and D-penicillamine (D-PA) as an anti-rheumatic drugs were orally administrated daily from days 0 to 20. On day 21, anti-OVA IgG2a and interferon (IFN)-gamma as indicators of Th1 responses and anti-OVA IgG1 and interleukin (IL)-10 as those of Th2 responses were measured. Treatments with IND, DEX, MTX and AUR were followed by decreases in OVA-specific IgG and proliferation of spleen cells to the antigen. Treatments with IND, DEX, MTX and AUR inhibited both Th1 and Th2 immune responses, although the inhibitory effects of these drugs on the antigen-specific IgG2a and IFN-gamma production appeared to be greater than those on IgG1 and IL-10 production. D-PA failed to influence anti-OVA IgG, IgG2a and IgG1 production as well as IFN-gamma and IL-10 secretion. Administrations of all the drugs used resulted in suppression of antigen (OVA)-induced arthritis in mice which was associated with inhibition of anti-OVA IgG2a but not IgG1 production. These results suggest that anti-arthritic drugs including IND, DEX, MTX and AUR appear to suppress Th1 and, to a lesser extent, Th2 immune responses, and their anti-inflammatory effects on human rheumatoid arthritis might be at least in part explained by downregulation by these drugs of Th1 responses involved in the disease.

Analysis of the human, bovine and rat 33-kDa proteins and cDNA in retina and pineal gland.

A monoclonal antibody (mAb) was produced against a bovine retinal 33-kDa protein. Several clones of 33-kDa protein were isolated from each library of cDNA from human, bovine and rat retinas and rat pineal gland by mAb screening and by hybridization with cDNA probes. Each of the four cDNA sequences was determined and amino acid (aa) sequences were deduced from the nucleotide sequences. The latter were nearly identical in rat retina and rat pineal gland (99.6%) and were similar in human, bovine and rat retina (more than 87%). Each of these cDNAs had one long ORF and encoded 245 or 246 aa. The deduced aa sequences in rat retina and rat pineal gland were virtually identical and the sequences in human, bovine and rat retina were highly homologous (more than 88%). The predicted Mr for each of these proteins was 28,246 in the human, 28,176 in bovine, 28,143 in rat retina, and 28,129 in rat pineal gland. Each of the sequences has a putative site for phosphorylation by A kinase; we have confirmed that the putative site is Ser73. These results show that the 33-kDa proteins in the retina and pineal gland have the same sequences and the same phosphorylation site and suggest that the functional role of this protein is the same in the retina and pineal gland.

The sequence of human retinal S-antigen reveals similarities with alpha-transducin.

The complete amino acid sequence of human retinal S-antigen (48 kDa protein), a retinal protein involved in the visual process has been determined by cDNA sequencing. The largest cDNA was 1590 base pairs (bp) and it contained an entire coding sequence. The similarity of nucleotide sequence between the human and bovine is approximately 80%. The predicted amino acid sequence indicates that human S-antigen has 405 residues and its molecular mass is 45050 Da. The amino acid sequence homology between human and bovine is 81%. There is no overall sequence similarity between S-antigen and other proteins listed in the National Biomedical Research Foundation (NBRF) protein data base. However, local regions of sequence homology with alpha-transducin (T alpha) are apparent including the putative rhodopsin binding and phosphoryl binding sites. In addition, human S-antigen has sequences identical to bovine uveitopathogenic sites, indicating that some types of human uveitis may in part be related to the animal model of experimental autoimmune uveitis (EAU).

Rat pineal S-antigen: sequence analysis reveals presence of alpha-transducin homologous sequence.

S-antigen (S-Ag) is a soluble, highly antigenic protein, the administration of which induces autoimmune uveitis. This protein is found in the retina and pineal. Retinal S-Ag from three species has been sequenced. In this study rat pineal S-Ag was sequenced. Clones were isolated from a rat pineal lambda gt11 cDNA library by probing with a 300 bp fragment of mouse retinal S-Ag cDNA containing the 5'-coding region. The largest clone isolated (RPS-118; 1364 bp) contained the entire coding sequence. Comparison of the rat pineal and mouse retinal S-Ag nucleotide sequences indicated a high homology (95%). The deduced amino acid sequence was found to contain 403 residues (congruent to 44 992 Da). Comparison of the rat pineal and mouse retinal S-Ag amino acid sequences also revealed high homology (97%). The similarity of both the nucleotide and amino acid sequences of rat pineal and mouse retinal S-Ag indicates that expression of the S-Ag gene in both tissues is similar. Further analysis of the rat pineal S-Ag sequence indicated that it contained essentially the same major uveitopathogenic region of S-Ag present in bovine retina; minor uveitopathogenic sites were somewhat different. As is true of retinal S-Ag, rat pineal S-Ag contains the same consensus phosphoryl-binding site present in many GTP/GDP-binding proteins and a homologous sequence found in the C-terminus of alpha-transducin. These sequences may play a role in the action of pineal S-Ag in transmembrane signal transduction.

Identification of autoreactive T cells in Vogt-Koyanagi-Harada disease.

PURPOSE: To determine the finer specificity and immunologic features of autoreactive T cells in Vogt-Koyanagi-Harada (VKH) disease. METHODS: T-cell clones (TCCs ) specific to tyrosinase family proteins were raised from the peripheral blood mononuclear cells (PBMCs) of patients with VKH disease, and the response of the TCCs to 30-mer peptides was determined. The TCCs that were reactive to the peptides with strong binding sites for HLA DRB1*0405 were initially tested. Then, a finer specificity of these TCCs against 12- to 14-mer peptides was determined. The cytokine production of these clones was measured by ELISA. RESULTS: A total of 62 stable TCCs were established from the PBMCs of five patients with VKH (28 clones against tyrosinase, 34 clones against tyrosinase-related protein [TRP]1). Five of 28 TCCs for tyrosinase and 2 of 34 for TRP1 were reactive to the 30-mer peptides with strong binding sites for HLA DRB1*0405. These seven clones showed proliferative responses to one or more of the 12- to 14-mer peptides that match the motif of the strong binding site for HLADRB1*0405. Five of seven of the TCCs may be T-helper (Th) type 1, one of the remaining TCCs may be Th0, and the other may be Th2. CONCLUSIONS: The autoreactive T cells against tyrosinase and/or TRP1 may contribute to the development of VKH disease.

Participation of protein kinases in staurosporine-induced interleukin-6 production by rat peritoneal macrophages.

The incubation of rat peritoneal macrophages in the presence of staurosporine, a non-specific protein kinase inhibitor, induced interleukin-6 (IL-6) production in a time- and concentration-dependent manner at 6.3-63 nM, but at 210 nM, the stimulant effect on IL-6 production was reduced. The levels of IL-6 mRNA as determined by a reverse transcription-polymerase chain reaction were also increased by staurosporine in parallel with the ability to induce IL-6 production. Compounds structurally related to staurosporine including K-252a (non-specific protein kinase inhibitor) and KT-5720 (inhibitor of cyclic AMP-dependent protein kinase, PKA), did not increase IL-6 production by peritoneal macrophages. Staurosporine-induced increases in IL-6 production and expression of IL-6 mRNA were decreased by the PKC inhibitors, H-7 (2.7-27 microM), Ro 31-8425 (1-10 microM) and calphostin C (0.3-3 microM) and by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002 (30-100 microM), but were further increased by the protein tyrosine kinase (PTK) inhibitor, genistein (12-37 microM). The staurosporine-induced increase in IL-6 production was not affected by the PKA inhibitor, H-89 (0.1-3 microM). These findings suggest that the induction of IL-6 production by staurosporine is secondary to elevation of IL-6 mRNA level, which, in turn, is positively regulated by the activation of PKC and PI 3-kinase and negatively regulated by the activation of PTK. PKA does not appear to play a significant role.

An approach for studies of mediator-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery.

1. Although intravital microscopy is the method of choice for observation of inflammatory leukocyte rolling and adhesion in small venules in vivo, a problem with this technique is that surgical exposure of suitable tissues per se triggers the rolling mechanism. In this study, we describe an approach to investigate induction of rolling in undisturbed microvessels. For this purpose, intravital microscopic observation of leukocyte rolling and adhesion in the rat mesentery was combined with histological determination of the intravascular concentrations of polymorphonuclear and mononuclear leukocytes (PMNL and MNL). 2. By relating the histologically determined number of intravascular leukocytes to either microvessel volume or to the erythrocyte concentration, the baseline MNL and PMNL content was found to be 3-6 fold higher in venules than in systemic blood. This increase in microvessel leukocyte concentration did not seem to be related to leukocyte-endothelium interactions, because the leukocyte concentration was similarly elevated in arterioles where rolling and adhesion did not take place. 3. Preparation of the rat mesentery for intravital microscopy time-dependently increased the venular PMNL concentration to over 100 fold the systemic PMNL concentration 45 min after exteriorization of the small intestine. The MNLs were much less responsive to the preparative manipulation. By treatment with the polysaccharide fucoidin (inhibits rolling but not firm adhesion per se), or by use of intravital microscopy immediately before tissue fixation, approximately 90% of the accumulated venular PMNLs were found to represent rolling cells. 4. Intraperitoneal injection of 10(-3) M histamine increased the venular PMNL (but not the MNL) concentration to almost 50 fold the systemic PMNL value. The histamine response did not vary with venular diameter, and the relative contribution of rolling vs firmly adherent cells to the PMNL, accumulation was again approximately 90%. Intraperitoneal injection of leukotriene C4, but not prostaglandin E2, caused a significant increase in venular PMNL concentration. 5. Systemic treatment with the anti-P-selectin monoclonal antibody PB1.3 had no effect on the histamine-induced venular PMNL accumulation (i.e. rolling) in female Wistar or male Sprague-Dawley rats. On the other hand, identical treatment with PB1.3 very effectively inhibited the histamine-induced PMNL response in the mesentery of rabbits. 6. In conclusion, we have shown that a histologically determined increase in leukocyte concentration in rat mesenteric venules may be used as an index of mediator-induced leukocyte rolling if the relative contribution of rolling and firm leukocyte adhesion is first determined, for example by the means described in this study. This relatively simple approach may be very useful for studying various aspects of leukocyte rolling when the 'spontaneous' rolling triggered by preparation of tissues for intravital microscopy is undesirable.