Expression Profiling of Human Basophils: Modulation by Cytokines and Secretagogues

<div><p>Human basophils are an accessible participant of the human allergic reaction. There is natural variation in various functional endpoints and in signaling molecule expression but there has been only a limited effort to place this information in the context of mRNA expression profiles. This study examined the hypothesis that unique mRNA signatures could be identified during the response of human basophils to several known forms of stimulation. Highly purified human basophils were cultured in vitro and exposed to IL-3, IL-5, NGF, IL-33, IL-2, anti-IgE Ab, or FMLP and the mRNA profiles examined by microarrays. The response to IL-3 and anti-IgE Ab were examined on 2–3 time frames and the response to IL-3 examined at several concentrations. In addition, the mRNA signatures of 3 different potential phenotypes were examined. These included basophils with the so-called non-releaser phenotype, and basophils from atopic and non-atopic subjects. Given the role of IL-3 in basophil maturation and the known profound effects on mature basophil function, it was not surprising that IL-3 showed the greatest influence on the basophil transcriptome. However, it also became apparent that the act of isolating and culturing basophils was sufficient to induce a large number of changes in the transcriptome, despite high viability and recovery. These “culture-effect” changes dominated the changes in mRNA profiles induced by other stimuli. Unique signatures for anti-IgE antibody and IL-33 could be identified although the number of gene transcripts (6–30) that were unique to these two stimuli was very limited. There were no apparent unique profiles for IL-5, NGF, IL-2 or FMLP. Therefore, a potential tool for screening basophil phenotypes was limited to changes that could be induced by IL-3 (or no IL-3), IL-33 and anti-IgE Ab.</p></div

FceRI density and spontaneous secretion from human basophils

<div><p>Mast cells and basophils depend on aggregation of the high-affinity IgE receptor, FceRI, to initiate secretion. A variety of studies have shown that FceRI densities vary 100 fold among subjects’ basophils and it has been speculated that high densities might be responsible for unusual behaviors of the cells, notably sensitivity to certain monomeric IgE antibodies or spontaneous release. These studies experimentally examined the density dependence of spontaneous release and signaling element expression in subjects’ basophils with FceRI densities ranging from approximately 6000 to 600,000 per cell. Through the use of numerical simulation, this study examined the expectations for spontaneous receptor aggregation and aggregate persistence at densities of FceRI ranging from 5000 to 500,000. Experimentally, FceRI density was not associated with greater spontaneous histamine release even when secretion was enhanced by the inclusion of deuterium oxide in the buffers. There was also no association of 15 activating or de-activating signaling elements with FceRI density. The numerical simulations demonstrated that at densities of ≈500,000 receptors, 13% of receptors were involved in transient aggregates at any given moment but that these aggregates rarely persisted for greater than 10 milliseconds. In contrast, a weak linear antigen aggregator, with ligand affinities known to induce secretion, would generate aggregates persisting for an average of ≈60 milliseconds. These results suggest that although a high density of FceRI likely produces a large number of transient aggregates, these aggregates do not persist long enough to induce signaling that results in secretion and do not induce the cells to alter their expression of several signaling elements known to be important in regulating secretion from human basophils. The results set some boundaries on the aggregation requirements for inducing histamine release from human basophils.</p></div

Changes in FceRI, syk or lyn expression during up-regulation of FceRI by culture for 4 days in 5 ng/ml IL-3 ± 1 µg/ml IgE.

<p>Panel A, peripheral blood basophils (n = 3). Panel B, CD34B starting on day 17 of culture (n = 4).</p

Concentration-dependence of the transcriptome response to IL-3.

<p>Panel A: Slope of SIL3-51T gene set for a 24 hour culture at the concentrations shown. The data points for each of the concentrations were derived from different numbers of experiments (n = 2 for 0.06 ng/ml, and n = 4 for 0.2, 2.0 and 10.0 ng/ml) and the plots represent composites of the results from different experiments. The lines represent the comparison with the +IL-3 (●) dataset or the noIL3 dataset (○). The square symbols represent the slopes for an IL-5 at 10 ng/ml exposure (relative to the SIL3-51T test set) and open triangles, the slope for NGF at 10 ng/ml). Panel B: Fold changes in three groups of transcripts. Two groups were derived from SIL3-51T, those that increased with IL-3 (●, n = 34) and those that decreased with IL-3 (■, n = 17). The third group (○) are transcripts that increase greater than 5 fold at the 10 ng/ml IL-3 concentration. Panel C: value of the SIL3-51T relationship between concentrations of IL-3. The ordinate is the slope of the SIL3-51T relationship for IL-3 concentration(i) vs. concentration(i+1) where (i) is a chosen concentration and (i+1) the next higher concentration, for example, 0.06 ng/ml vs 0.2 ng/ml (or 0.2 vs. 2.0, 2.0 vs. 10). The two lines are the slopes for the noIL3 relationship (○) and the +IL3 relationship (●). Panel D: Concentration dependence for two subsets of transcripts chosen with a hierarchical clustering algorithm (see text), those that show similar changes at 2 vs. 10 ng/ml (●) and those that show significant differences at the two concentrations (○).</p

A single example of FceRI expression vs. syk expression (in units of Western blot standard).

<p>A single example of FceRI expression vs. syk expression (in units of Western blot standard).</p

Representation of an “over-head” view of receptor density and associations.

<p>Each “dot” represents the radius of gyration of a surface IgE molecule. Panel A; density of 5000/cell. Panel B; density of 500,000/cell. Panel C; density of 500,000/cell where green dots represent IgE-FceRI molecules in close enough proximity to be considered a potentially functional aggregate based on published studies.</p

Design of a signature algorithm that incorporates the results for the various stimuli.

<p>The top half shows the algorithm that was explored using the existing information and the bottom half the algorithm pruned for those elements that were found to be consistent with the results.</p

Heatmaps of selected genes to be considered as signatures for stimulation by IL-3, IL-33 and anti-IgE Ab.

<p>Seven conditions are presented across the horizontal and 6 potential signature sets along the vertical. There are 24, 7, 4, 6, 34 and 16 genes in the 6 signature sets (see Table A3 in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126435#pone.0126435.s001" target="_blank">S1 appendix</a> for a textual listing). The IL-3 conditions include 10 and 2 ng/ml IL-3 for 24 hrs and no IL-3 for 24 hrs. IL-33 at 20 ng/ml for 24 hours and anti-IgE Ab at its optimal concentration of 0.5 μg/ml for 2, 15 and 72 hrs are also shown. The 72 hour incubation for anti-IgE includes IL-3 at 10 ng/ml and the comparison is between IL-3 alone for 72 hours vs. IL-3 plus anti-IgE Ab for 72 hours. All other comparisons are between the stimulation condition and freshly isolated cells (Day 0). The insert shows that the gradient of colors progress from blue for down-regulation of expression to green for no change and red for up-regulation of expression in the range of 0.1 to 10 fold. Values below 0.1 or above 10.0 are blue and red respectively. The signature set labeled ‘IL-3 inversion set’ refers to the genes whose expression behave in opposite directions in the presence or absence of IL-3 and represent a subset of SIL-51T. The set labeled ‘hi IL-3’ are genes that are changed only at 10 ng/ml IL-3 while the set labeled ‘mid IL-3’ are selected from genes that change to a similar extent in both 2 and 10 ng/ml IL-3. As noted in the text, there a 2 potential sets for stimulation with anti-IgE Ab, reflecting differences between 2 and 15 hours of stimulation.</p

Pearson R for each element vs. FceRI; all p>0.05.

<p>Pearson R for each element vs. FceRI; all p>0.05.</p

Summary of correlations for FceRI density vs. spontaneous release.

<p>Summary of correlations for FceRI density vs. spontaneous release.</p