Levels of tryptase, chymase, and FcϵRIα messenger RNA in human skin are unchanged after IgE-dependent stimulation of cutaneous mast cells in vivo
Authors: Han-Zhang Xiaa, Burton Zweiman, Lawrence B. Schwartz
Journal: Journal of Allergy and Clinical Immunology (1997)

IgE-dependent activation of mast cells is associated with secretion of tryptase and chymase, along with other granule mediators, and internalization of the high-affinity receptor for IgE, FcϵRI. We hypothesized that messenger RNA levels in mast cells corresponding to these molecules might be dramatically elevated within 24 hours of mast cell activation in preparation for replacement of lost proteins. Ultrastructural examinations of human lung-derived mast cells within 24 hours of activation show increased smooth and rough endoplasmic reticulum,1Dvorak AM Schleimer RP Lichtenstein LM Human mast cells synthesize new granules during recovery from degranulation: in vitro studies with mast cells purified from human lungs.Blood. 1988; 71: 76-85PubMed Google Scholar providing support for our hypothesis. Consequently, 3 mm punch biopsy specimens were taken from anesthetized (with 2% lidocaine) skin sites 6 hours and 24 hours after intradermal injection (0.03 ml) of either an antigen (≥rass or ragweed extract, 100 PNU/ml; Greer Laboratories, Lenoir, N.C.) to which the subject was sensitive or a diluent control as previously described.2Vowels BR Rook AH Cassin M Zweiman B Expression of interleukin-4 and interleukin-5 mRNA in developing cutaneous late-phase reactions.J Allergy Clin Immunol. 1995; 96: 92-96Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Each biopsy specimen was immediately snap-frozen in liquid nitrogen. Total RNA was extracted by use of RNAzol B (Tel-Test, Inc., Friendswood, Texas), a modified guanidinium thiocyanate technique,3Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar as described by the manufacturer. Each sample of target RNA was subjected to competitive reverse-transcription polymerase chain reaction (RT-PCR) with gene-specific primers and competitor molecules consisting of complementary RNA for tryptase, chymase, the α subunit of FcϵRI, and glyceraldehyde-3-phosphate dehydrogenase (≥3PDH), as described previously for tissue-derived mast cells and peripheral blood basophils.4Xia H-Z Kepley CL Sakai K Chelliah J Irani A-MA Schwartz LB Quantitation of tryptase, chymase, FcεRIα, and FcεRIgamma mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction.J Immunol. 1995; 154: 5472-5480PubMed Google Scholar The cRNA competitor molecules were generated from corresponding complementary DNAs that had small portions of the sequence deleted from a region internal to the primers. The use of cRNA competitor molecules at the RT step corrects for variability in the efficiency of RT and of PCR in different experiments. A ratio (R) of target to competitor PCR product was calculated, and this represents an index of the relative amounts of mRNA. The molar concentration of cRNA equals the concentration of mRNA in the RT-PCR reaction mixture when the R value is equivalent to a molar target-to-competitor ratio of 1. To determine target mRNA concentrations when a limiting amount of cellular RNA is available, R n values were first determined at a single competitor cRNA concentration (C n). A C 1 value was next calculated for the R 1 value corresponding to a target-to-competitor molar ratio of 1. R 1 values were 1.32 for tryptase, 1.58 for chymase, 1.89 for FcϵRIα, and 1.35 for G3PDH. C 1 was calculated according to the following formula: where K is the slope constant corresponding to a particular molecule and to a set of reaction conditions as calculated from the following formula: log(R) = K × log(C) + B As described previously,4Xia H-Z Kepley CL Sakai K Chelliah J Irani A-MA Schwartz LB Quantitation of tryptase, chymase, FcεRIα, and FcεRIgamma mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction.J Immunol. 1995; 154: 5472-5480PubMed Google Scholar log(R) shows a linear relationship to log(C) with a slope K and a y-axis intercept B. K values (mean ± SD) determined in separate experiments4Xia H-Z Kepley CL Sakai K Chelliah J Irani A-MA Schwartz LB Quantitation of tryptase, chymase, FcεRIα, and FcεRIgamma mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction.J Immunol. 1995; 154: 5472-5480PubMed Google Scholar with mRNA from HMC-1 cells, KU812 cells, peripheral blood basophils, and tissue mast cells dispersed from lung and skin were as follows: tryptase, -1.24 ± 0.17, n = 12; chymase, -1.21 ± 0.11, n = 6; FcϵRIα, -1.05 ± 0.12, n = 6; and G3PDH, -1.24 ± 0.25, n = 19. Values for tryptase, chymase, and FcϵRIα were then normalized to the corresponding value for G3PDH to obtain the relative amount of each mRNA molecule in the skin biopsy specimen. This latter step diminishes the variability arising from differences in the cellular RNA content of different biopsy specimens. Each of six atopic subjects (four men) with quiescent respiratory allergic diseases and no skin disease were challenged by intradermal injection of allergen (100 PNU/ml) at two sites and of diluent at two sites. Wheal (12 to 25 mm) and flare (31 to 50 mm) responses occurred at each allergen site 20 minutes after challenge with antigen, whereas buffer-challenged sites at this time point showed 0 to 3 mm wheals and no flares. Induration (>10 mm diameter) was observed in all allergen-challenged sites 6 hours later and in three of the six allergen-challenged sites at 24 hours. No buffer-challenged site exhibited detectable induration (≥1 mm) at either time point. After RNA extraction, relative mRNA levels of chymase, tryptase, and FcϵRIα were determined as shown in Fig. 1. No mRNA was detected in the 6-hour buffer control specimen in one subject, which suggested that RNA had been degraded. This data point was not considered in the analyses. Also, no RT-PCR product for target or competitor chymase nucleic acid was found in one 24-hourbuffer-challenged site, and this data point was also deleted from the analyses. Because of limiting quantities of RNA, each assay could be performed only once. Although there was a tendency for FcϵRIα mRNA levels to increase at the 24-hour antigen-challenge site compared with the buffer-challenge site and for the tryptase mRNA levels to decrease at the 6-hour antigen-challenge site compared with the buffer-challenge site, no statistically significant differences in the median values between antigen-challenge and buffer-challenge sites were found (p > 0.2; Wilcoxon signed-rank test for nonparametric data). Also, there was no significant correlation between levels of mRNA for the granule mediators (tryptase and chymase) and mRNA of the membrane receptor (FcRIα) in the biopsy specimens. In this study we found highly variable amounts of mRNA for tryptase, chymase, and FcϵRIα in the skin of different subjects. This variability was also found in a previous study of mast cells dispersed from skin.4Xia H-Z Kepley CL Sakai K Chelliah J Irani A-MA Schwartz LB Quantitation of tryptase, chymase, FcεRIα, and FcεRIgamma mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction.J Immunol. 1995; 154: 5472-5480PubMed Google Scholar In part, this may reflect variations in the relative mast cell content in tissue samples taken from different sites, as suggested by the variations in mRNA content of the 6-hour and 24-hour buffer-challenge sites (assumed to be unstimulated) in the same individual. Also, as found previously in studies with dispersed mast cell preparations in which data could be normalized to the number of mast cells, mRNA content per mast cell varies from individual to individual. Finally, the possibility that basophils were recruited into the allergen-challenge sites by 24 hours and influenced mRNA levels should be considered. Although FcϵRIα mRNA levels in mast cells and basophils are comparable, tryptase mRNA levels are 105 times lower in basophils than in mast cells, and chymase mRNA is undetectable in basophils.4Xia H-Z Kepley CL Sakai K Chelliah J Irani A-MA Schwartz LB Quantitation of tryptase, chymase, FcεRIα, and FcεRIgamma mRNAs in human mast cells and basophils by competitive reverse transcription-polymerase chain reaction.J Immunol. 1995; 154: 5472-5480PubMed Google Scholar FcϵRIα mRNA levels were higher in five of six allergen-challenged sites than in corresponding buffer-challenged sites at 24 hours. Previous studies of allergen challenge in which the skin-chamber technique was used suggested that basophils may be recruited into these sites as early as 1 hour after challenge.5Atkins PC Schwartz LB Adkinson NF von Allmen C Valenzano M Zweiman B In vivo antigen-induced cutaneous mediator release: simultaneous comparisons of histamine, tryptase, and prostaglandin D 2 release and the effect of oral corticosteroid administration.J Allergy Clin Immunol. 1990; 86: 360-370Abstract Full Text PDF PubMed Scopus (50) Google Scholar On the other hand, the FcϵRIα R1 values at the 24-hour antigen-challenge sites were no higher than those at the 6-hour allergen- and buffer-challenge sites. Thus it is unlikely that basophils substantially affected the cellular levels of FcϵRIα mRNA under the conditions of this study. We conclude from these data that mRNA levels for tryptase, chymase, and FcϵRIα do not change substantially in mast cells in vivo within 24 hours of allergen-mediated activation. This suggests that increases in mRNA levels for these molecules are not likely required to replenish depleted stores of these proteins in mast cells recovering from degranulation. Instead, translation and posttranslational processing may be more important. One consideration in this analysis is the percentage of mast cells actually activated in vivo by the allergen challenge. For example, if only a small percentage of mast cells degranulated (a situation we consider unlikely), a consistent increase in mRNA levels among these cells might be masked by the mRNA levels in the larger numbers of unstimulated mast cells. Another consideration is mRNA turnover. mRNA content at any one time reflects the result of both synthesis and degradation. Therefore our findings do not exclude the possibility that synthesis of new mRNA for these proteins increases after mast cell activation but do suggest that the quantity of new mRNA does not substantially raise the concentration of that particular mRNA, perhaps because mRNA degradation increases proportionately.