René E. Déry

Inhibition of Allergic Inflammation in the Airways Using Aerosolized Antisense to Syk Kinase

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of FcγR and FcεR, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of β2 integrins, α4 integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.

Frontline: Inhibition of allergen-induced pulmonary inflammation by the tripeptide feG: a mimetic of a neuro-endocrine pathway.

Interactions between the neuro‐endocrine system and immune system help maintain health. One interaction involves the superior cervical ganglia (SCG), which regulate the prohormone submandibular rat 1 (SMR1) produced by the submandibular gland (SMG). A peptide derived from SMR1, feG, has anti‐inflammatory activity, and modification to D‐isomer feG enhances bioactivity. We tested feG as a therapeutic agent for airways inflammation, using rats sensitized by OVA or Nippostrongylus brasiliensis (Nb). Treatment with feG but not fdG down‐regulated OVA‐challenge‐induced increases in bronchoalveolar lavage (BAL)‐derived macrophages, eosinophils and PMN (neutrophils) by 44%, 69% and 67%, respectively, at 24 h. We found that feG also reduced ICAM‐1 on BAL‐derived macrophages and eosinophils by 27% and 65%, and L‐selectin on PMN by 55% following OVA challenge. Furthermore, feG but not fdG reduced the OVA‐induced TNF increase in BAL fluid. We showed that feG also down‐regulated both hyper‐responsiveness to methacholine (by 27%) and microgranulomata formation in the lung parenchyma. In Nb‐challenged rats, feG treatment inhibited ex vivo allergen‐induced contraction of tracheal smooth muscle by up to 73%. In conclusion, feG, which is a mimetic of a peptide derived from a rat salivary gland prohormone, has anti‐inflammatory properties in allergic airways inflammation in Brown‐Norway rats. The role of the SCG‐SMG neuro‐endocrine pathway in allergic asthma and other inflammatory diseases requires additional study.

Inhibition of Allergic Inflammation by C-Terminal Peptides of the Prohormone Submandibular Rat 1 (SMR-1)

Background: The C-terminal of the prohormone submandibular rat 1 protein (SMR-1) contains several small peptides that reduce the severity of allergic inflammation and septic shock, and are part of the cervical sympathetic trunk-submandibular gland (SMG) axis of neuroendocrine immunology. These peptides include the heptapeptide, submandibular gland peptide-T and the tripeptide FEG. The D-isomeric form of this tripeptide, feG, which is active when administered orally, reduces LPS-provoked leukocyte rolling on mesenteric venules and influx of inflammatory cells into the peritoneum and intestinal muscle. Methods: To investigate the mechanism of action of these peptides, the influx of inflammatory molecules into the airways, and several properties of human neutrophils were examined. Results: Oral feG (1 mg/kg) inhibited the influx of inflammatory cells into the airways lumen of allergen challenged, sensitized Brown Norway rats. This inhibition occurred whether feG was given 30 min prior to 6 h post allergen challenge. Moreover, feG in picomolar to nanomolar concentrations inhibited PAF elicited chemotaxis by 30–40%, but the peptides did not affect superoxide production or phagocytosis by neutrophils. feG reduced PAF-stimulated expression of CD11b. Conclusions: feG may exert its anti-inflammatory effects by modulating the expression and functions of β2 integrins. The CST-SMG axis may be a major neuroendocrine pathway that modulates allergic asthma and other inflammatory responses.

Regulation of CD8 Expression in Mast Cells by Exogenous or Endogenous Nitric Oxide

We recently reported a novel CD8 molecule on rat alveolar macrophages and peritoneal mast cells (PMC). However, little is known about the regulation of CD8 expression and function on these cells. We investigated the regulation of CD8 expression on PMC by NO, because NO can regulate inflammatory responses and also because anti-CD8 Ab stimulates inducible NO synthase and NO production by PMC and alveolar macrophages. Ligation of CD8α on PMC with Ab (OX8) induced CD8α mRNA expression after 3–6 h, and flow cytometry demonstrated that OX8 treatment increased CD8α protein expression compared with PMC treated with isotype control IgG1. To test whether NO mediates the up-regulation of CD8α, we used the NO donor S-nitrosoglutathione (500 μM) and NO synthase inhibitors (NG-monomethyl-l-arginine and NG-nitro-l-arginine methyl ester; 100 μM). S-nitrosoglutathione up-regulated both mRNA and protein expression of CD8α in PMC compared with that in sham-treated cells, while NO synthase inhibitors down-regulated OX8 Ab-induced CD8α expression. To investigate how NO regulates CD8 expression on PMC, we examined the cGMP-dependent pathway using 8-bromo-cGMP (2 mM) and the guanylate cyclase inhibitor, 1H-oxadiazoloquinoxalin-1-one (20 μM). 8-Bromo-cGMP up-regulated CD8 expression, whereas 1H-oxadiazoloquinoxalin-1-one down-regulated its expression. Thus, ligation of CD8 up-regulates CD8 expression on PMC, a response mediated at least in part by NO through a cGMP-dependent pathway. The significance of this up-regulation of CD8α on mast cells (MC) is unclear, but since ligation of CD8 on MC with OX8 Ab can alter gene expression and mediator secretion, up-regulation of CD8 may enhance the MC response to natural ligation of this novel form of CD8.

Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages

Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrophages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mast cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alveolar macrophages (NR8383) were studied for the detection of mRNA for beta-actin, TNF-alpha and/or CD8alpha. Each type of cell has unique optimal conditions for RT-in situ PCR. The following parameters were carefully evaluated for optimization: protease digestion, DNAse digestion, heparinase digestion, RT, PCR cycle number and signal development with chromagen. Heparinase digestion was required for PMC mRNA detection because they contain large amounts of heparin proteoglycan, which is a potent inhibitor of RT and Taq polymerase enzymes. Only a few PCR cycles were needed to produce a cytoplasmic signal for mRNA transcripts in RCMC, whereas other types of cells (PMC, BALC and NR8383) needed at least 20 cycles for mRNA detection. The mRNA signal in PMC was localized to the perinuclear region, whereas mRNA in other cell types (RCMC, BALC and NR8383) were detected throughout the cytoplasm. Furthermore, modified Southern blot analysis for TNF-alpha in RCMC treated with RT-in situ PCR demonstrated the specificity of amplification product. The modified and optimized protocols for this procedure were successfully applied to detect and localize several mRNA transcripts in rat MC and AM. The approach is valuable and can be used to further study selected gene expression in these and other cell types.

Immunomodulation by the submandibular gland

Abstract We have established that decentralization (cutting sympathetic nerve trunk) of the superior cervical ganglia bilaterally reduces the magnitude of allergic inflammation in the airways of rats. The magnitude of anaphylactic and endotoxic hypotension, and of gastrointestinal inflammation was also reduced. This anti-inflammatory activity was dependent upon intact submandibular glands. Reconstitution of sialadenectomized (removal of submandibular glands) rats with soluble extracts of the submandibular glands identified two polypeptides with anti-inflammatory activities. The sequences of these polypeptides were found within a prohormone, submandibular gland rat 1 (SMR1). The C-terminal peptide TDIFEGG, has been studied extensively. Using sequential amino acid substitutions and systematic removal of C terminal or N terminal amino acids, we established that the tripeptide FEG is biologically active. Modification of FEG to the D-isomeric feG, enhances its activity in some assay systems. We postulated that feG would inhibit airways inflammation, and tested this using a model ofallergic asthma, namely the Brown Norway rat sensitized to ovalbumin (OA). Sensitized rats were challenged 14 to 21 days later with aerosolized OA. This challenge markedly increased numbers of inflammatory cells recovered from the airways after 24 hour (29×10 6 , n=23) compared to saline controls (1×10 6 , n=4). The infiltrating cells included macrophages (10×10 6 ), neutrophils (9×10 6 ) and eosinophils (9×10 6 ). Intravenous (0.25 mg/kg) or oral feG (1 mg/kg) given 30 min prior to OA significantly inhibited influx of inflammatory cells by 50 to 70%. feG reduced inflammatory cell infiltration when given 30 min before to 3 to 6 hour post allergen exposure. Oral feG reduced the numbers of macrophages, neutrophils and eosinophils. One of the mechanisms underlying the effects of feG may be its ability to inhibit PAF-induced expression of CD11b on purified human neutrophils. It is possible that feG may be useful in the treatment of allergic asthma, given either as an oral prophylactic, or as a post exposure therapeutic. The cervical sympathetic nerve trunk-submandibular gland axis of neuroendocrine regulation of inflammation may be dysfunctional in inflammatory diseases and provide opportunities for new therapeutic intervention. This axis may be sensitive to modulation by central and peripheral neural mechanisms that influence its function/dysfunction.