Effect of topical capsaicin on the cutaneous responses to inflammatory mediators and to antigen in man

Topical capsaicin pretreatment is known to deplete cutaneous sensory nerves of neuropeptides. We have assessed the effect of topical capsaicin pretreatment on the responses to intradermal injections of histamine and platelet-activating factor (PAF) in six normal subjects, and of prostaglandin E2, histamine, and antigen in 10 atopic subjects. Capsaicin pretreatment caused significant inhibition of the immediate flare response to histamine in both normal (19.8 +/- 2.6 to 7.3 +/- 2.9 cm2 at 5 minutes; p less than 0.01) and atopic subjects (16.5 +/- 1.4 to 10.3 +/- 1.9 cm2 at 5 minutes; p less than 0.01). The PAF-induced flare was also inhibited from 12.2 +/- 2.9 to 2.7 +/- 1.6 cm2 at 5 minutes after injection (p less than 0.01). In contrast, capsaicin pretreatment did not significantly alter the flare responses to prostaglandin E2 or antigen in atopic subjects. The acute wheal responses to all stimuli were unchanged, as was the late-phase response to antigen. These results support the hypothesis that the cutaneous vasodilator effect of histamine and PAF may be mediated by a local axon reflex involving the release of neuropeptides from sensory nerves. A consistent effect of capsaicin pretreatment on the flare response induced by endogenous mediators released during a cutaneous IgE-mediated response was not observed. Increases in microvascular permeability and the late-phase response to antigen are independent of neuropeptide release from cutaneous nerves.     

Sepsis-induced SOCS-3 expression is immunologically restricted to phagocytes

We have shown that immune cells from septic mice exhibit a suppressed response to exogenous stimuli in vitro. The suppressors of the cytokine signaling (SOCS) family are proteins that block intracellular signaling and can be induced by inflammatory mediators. Therefore, we hypothesized that SOCS-3 is up-regulated in immune cells in response to a septic challenge induced by cecal ligation and puncture (CLP). Mice were subjected to CLP or sham-CLP, and 2-48 h later, the blood, thymus, spleen, lung, and peritoneal leukocytes were harvested and examined. SOCS-3 was undetectable in thymocytes or blood leukocytes. In contrast, SOCS-3 was up-regulated in the spleen, lung, and peritoneal leukocytes in a time-dependent manner. Further examination revealed that only the macrophages and neutrophils expressed SOCS-3. These data suggest that cytokines and bacterial toxins present during sepsis have the ability to suppress the cytokine and/or lipopolysaccharide response and the function of immune cells by up-regulating SOCS-3.     

Altered patterns of migration of cytokine-producing T lymphocytes in skin-grafted naive or immune mice following in vivo administration of anti-VCAM-1 or -ICAM-1.

Naive or preimmunized (to B10.BR or BALB.k) C3H/HeJ mice received skin grafts from multiple minor histoincompatible B10.BR or BALB.k mice following antigen-specific portal venous (p.v.) pretransplant transfusion, a protocol known to produce prolongation of graft survival in naive animals. In addition, groups of mice received intravenous (i.v.) infusion following transplantation with a mixture of monoclonal antibodies (mAb) to vascular adhesion molecule-1L: very late activation antigen-4 (VCAM-1:VLA-4) or intracellular adhesion molecule-1:lymphocyte function-associated antigen-3 (ICAM-1:LFA-1). Cells were harvested from different tissues of the grafted mice at various times post grafting. RNA was extracted and analysed, using polymerase chain reaction, for expression of different cytokines potentially involved in the regulation of graft rejection [interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumour necrosis factor-alpha, interferon-gamma and transforming growth factor-beta]. In addition, using limiting dilution analysis, we investigated the frequency of allo-specific and third-party reactive cells producing IL-2 and IL-4 in vitro in different tissues of grafted mice following these treatments. The mAb treatment protocol which produced optimum increases in graft survival in naive versus immune mice was different, with anti-LFA-1:ICAM-1 superior for naive mice compared with anti-VLA-4:VCAM-1, and vice versa for immune animals. However, in each case, increased survival was associated with increases local to the graft in the frequency of occurrence of antigen-specific type-2 cytokine-producing cells.     

Induction of human airway smooth muscle apoptosis by neutrophils and neutrophil elastase

Neutrophils are an important component of airway inflammation and may interact with human airway smooth muscle cells (HASMC). We investigated the effect of neutrophils and of neutrophil-derived proteases on HASMC survival. When co-incubated with neutrophils (0.1-1 x 10(6) cells/ml), attachment of human ASMC was reduced to 12.3 +/- 4.3% compared with untreated controls after 72 h. HASMC showed nuclear condensation and fragmentation (41.6 +/- 8.1% compared with baseline of 3.1 +/- 0.4%), and the biochemical markers of apoptosis, annexin V binding (9.7 +/- 0.7%; baseline 1.1 +/- 0.3%) and cleaved caspase-3 expression, were observed. The proteolytic activity released by neutrophils was essential for the proapoptotic effect because inhibition of elastase activity by alpha(1)-antitrypsin and MeOSuc-Ala-Ala-Pro-Ala-CMK (MSACK) reduced HASMC apoptosis. Human neutrophil elastase (0.1-3 microg/ml) induced apoptosis of HASMC, as well as other neutrophil serine proteases, cathepsin G, and proteinase 3. Fibronectin degradation products were present in HASMC supernatants exposed to neutrophil-conditioned media and to neutrophil elastase. The local release of proteases from neutrophils present in airway smooth muscle cells may lead to HASMC apoptosis as a result of matrix degradation and loss of cell attachment. This may limit pathologic changes such as ASMC hyperplasia and extracellular matrix deposition seen in airway remodeling.     

Rapid, accurate genotyping of the common -alpha(4.2) thalassaemia deletion based on the use of denaturing HPLC

AIMS: To develop an alternative assay for specific genotyping of the -alpha(4.2) thalassaemia deletion based on the DNA sequence features surrounding the breakpoint. METHODS: The 5' and 3' ends of the breakpoint regions of the -alpha(4.2) allele and the normal homologous segments were sequenced in Chinese individuals. A sequence haplotype composed of four single nucleotide variations within the X2/X1 box of the -alpha(4.2) breakpoint region was found in all of the 10 Chinese -alpha(4.2) thalassaemia alleles studied. Based on these findings, a novel polymerase chain reaction (PCR)/denaturing high performance liquid chromatography (DHPLC) assay was developed for rapid genotyping of the -alpha(4.2) allele instead of traditional Southern blotting or Gap-PCR. This method involves amplification of the alpha globin target sequence encompassing these four polymorphic sites, followed by a partially denaturing HPLC analysis using the transgenomic WAVE DNA fragment analysis system. RESULTS: The three major genotypes (-alpha4.2/alphaalpha, -alpha(4.2)/--SEA, and alphaalpha/alphaalpha) could be distinguished through the characteristic chromatograms generated by the WAVE system. The accuracy of this technique was evaluated blindly, and the results were 100% (40 of 40) concordant with the genotypes previously characterised by Southern blotting or Gap-PCR. CONCLUSIONS: This study validates the PCR/DHPLC approach as a simple, rapid, highly accurate, and cost effective method, potentially adaptable for use in epidemiological surveys, genetic screening, and diagnosis of silent alpha+ thalassaemia and Hb H disease.     

Inhibition of inducible nitric oxide synthase expression by interleukin-4 and interleukin-13 in human lung epithelial cells.

Oral feeding of proteins causes peripheral T-cell tolerance, as revealed by reduced delayed-type hypersensitivity (DTH) reactivity after immunization. This type of tolerance can be due both to passive T-cell anergy and active immunosuppression. Using ovalbumin-fed mice we studied whether putatively immunostimulatory cytokines could break this state of mucosal tolerance. Cytokines were administered locally at the site of attempted sensitization. It was found that neither interleukin-2 (IL-2), interferon-gamma (IFN-gamma) nor granulocyte-macrophage colony-stimulating factor (GM-CSF) could restore the response to immunization. In contrast, local administration of IL-12 at the site of attempted immunization resulted in full recovery of DTH reactivity. The dichotomy between the two Th1 stimulatory cytokines IFN-gamma and IL-12 was also reflected by different effects on ovalbumin-specific antibody isotypes. Although both IFN-gamma and IL-12 downregulated serum IgG1-levels in tolerant mice, suggesting decreased ovalbumin-specific Th2 function, only local administration of IL-12 led to increased serum IgG2a levels. These results support the view that potentiation of Th1 effector function is critical for reversal of mucosal tolerance.