Pitfalls in the characterization of small intestinal lymphocytes

The morphological characteristics of human intestinal lymphocytes may vary depending upon whether they are examined in tissue sections or after isolation. This study uncovered some causes of these discrepancies. The majority of intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) are of the same size and granularity as peripheral blood mononuclear cells (PBMC), negating the idea that most IEL are large granular lymphocytes. LPL were previously shown to express markers of early activation by flow cytometry but not by immunohistochemistry. This study shows that it is the low density of surface antigen expression that explains this discrepancy: only the highly sensitive flow cytometric analysis can detect such low-density expression. Previous reports of intestinal macrophage and B cell numbers and functions vary markedly. This may be due to variable amounts of contamination with epithelial cells (EC) that lose their characteristic columnar appearance and cytoplasmic mucin upon isolation. They share many features of macrophages (such as morphology and binding to plastic and latex beads) but lack phagocytic ability and superoxide generation, explaining, perhaps, the wide variation in the reported functions of presumed intestinal macrophages. In addition, a large fraction of EC non-specifically strain with anti-immunoglobulin antibodies. Their contamination of intestinal lymphocyte preparations accounts for the discrepancy in B cell numbers identified by surface immunoglobulin expression versus B cell markers. Intestinal lymphocytes do indeed differ in many ways from circulating lymphocytes, but investigators must be attentive to factors that may artifactually alter their results.