IgG4 Immunostaining and Its Implications in Orbital Inflammatory Disease

<div><p>Objective</p><p>IgG4-related disease is an emerging clinical entity which frequently involves tissue within the orbit. In order to appreciate the implications of IgG4 immunostaining, we analyzed gene expression and the prevalence of IgG4- immunostaining among subjects with orbital inflammatory diseases.</p><p>Methods</p><p>We organized an international consortium to collect orbital biopsies from 108 subjects including 22 with no known orbital disease, 42 with nonspecific orbital inflammatory disease (NSOI), 26 with thyroid eye disease (TED), 12 with sarcoidosis, and 6 with granulomatosis with polyangiitis (GPA). Lacrimal gland and orbital adipose tissue biopsies were immunostained for IgG4 or IgG secreting plasma cells. RNA transcripts were quantified by Affymetrix arrays.</p><p>Results</p><p>None of the healthy controls or subjects with TED had substantial IgG4 staining. Among the 63 others, the prevalence of significant IgG4-immunostaining ranged from 11 to 39% depending on the definition for significant. IgG4 staining was detectable in the majority of tissues from subjects with GPA and less commonly in tissue from subjects with sarcoidosis or NSOI. The detection of IgG4+ cells correlated with inflammation in the lacrimal gland based on histology. IgG4 staining tissue expressed an increase in transcripts associated with inflammation, especially B cell-related genes. Functional annotation analysis confirmed this.</p><p>Conclusion</p><p>IgG4+ plasma cells are common in orbital tissue from patients with sarcoidosis, GPA, or NSOI. Even using the low threshold of 10 IgG4+ cells/high powered field, IgG4 staining correlates with increased inflammation in the lacrimal gland based on histology and gene expression.</p></div

Case demographics.

<p>*Ages are not available for 4 subjects.</p><p>Case demographics.</p

IgG4 status is independent of age and gender.

<p>*Ages are not available for 4 subjects.</p><p>IgG4 status is independent of age and gender.</p

Examples of gene expression differences comparing IgG4+ to IgG4- lacrimal gland tissue from subjects with NSOI, GPA, or sarcoidosis.

<p>Probe sets were selected for expression increases of more than 2.1 fold or decreases of more than 1.5 fold, p values <0.5, and sample annotation. A relevant Gene Ontology Biological Process (<a href="http://geneontology.org/" target="_blank">http://geneontology.org/</a>) is listed for each gene.</p><p>Examples of gene expression differences comparing IgG4+ to IgG4- lacrimal gland tissue from subjects with NSOI, GPA, or sarcoidosis.</p

Examples of gene expression differences comparing IgG4+ to IgG4- orbital tissue from subjects with NSOI, GPA, or sarcoidosis.

<p>Probe sets were selected for expression increases or decreases and ample annotation. A relevant Gene Ontology Biological Process (<a href="http://geneontology.org/" target="_blank">http://geneontology.org/</a>) is listed for each gene.</p><p>Examples of gene expression differences comparing IgG4+ to IgG4- orbital tissue from subjects with NSOI, GPA, or sarcoidosis.</p

Lacrimal tissues from NSOI patients with at least 10 IgG4+ PC/hpf show increased fibrosis and inflammation.

<p>IgG4+ orbit tissues lack the lowest fibrosis and inflammation scores. Each symbol represents the score for one subject. P-values are based on Mann-Whitney U-test.</p

A minority of subjects with inflamed orbits have markedly high IgG4+PC counts.

<p>The number of subjects in each category is shown.</p><p>A minority of subjects with inflamed orbits have markedly high IgG4+PC counts.</p

Principal component analysis based on significantly up-regulated and significantly down-regulated probe sets in TED orbital adipose (T) against uninflamed controls (C).

<p>The discovery set (Set 1) had 36 significantly up-regulated and 254 significantly down-regulated probe sets. The validation set (Set 2) had 66 significantly up-regulated and 604 significantly down-regulated probe sets. At least 1.5-fold change with FDR adjusted p-value < 0.05 is considered statistically significant.</p

Ages for each experimental group.

<p>*one repeated from set 1;</p><p>**two repeated from set 1</p><p>Ages for each experimental group.</p

Probe sets with a significantly lower signal in orbital tissue from TED subjects compared to uninflamed controls.

<p>Probe sets with a significantly lower signal in orbital tissue from TED subjects compared to uninflamed controls.</p