Isolation, maturational level, and functional capacity of human colon lamina propria plasma cells

Abstract

Background and aims: Large numbers of plasma cells (PC) localise in the intestinal lamina propria (LP) where they play a critical role in the defence against pathogens. This study analyses the level of maturation reached by normal human colon LPPC in comparison with that of bone marrow (BM) PC.

Methods: A technique was designed to purify LPPC by combining collagenase digestion of the mucosal layer and immunomagnetic selection of CD54(+) LP cells. It provided highly purified PC, as demonstrated by morphology, CD38(h) phenotype, and cytoplasmic IgA staining criteria. This procedure allowed comparison of in vitro functional capacities and a broad phenotypic analysis of BMPC and LPPC.

Results: LPPC and BMPC exhibited identical expression of differentiation markers (CD19(-/+), CD20(-), HLA-DR(low/-), VS38c(high)), survival molecules (CD95 (low/-), Bcl-2(+)), and B cell transcription factor profile, as well as similar in vitro Ig secreting kinetics (14 days) and lack of susceptibility to apoptosis by CD95 ligation. In contrast, they markedly differed in adhesion molecule expression, as LPPC showed higher levels of CD44 and CD21 and were alpha 4 beta 7(+) whereas BMPC lacked this integrin and expressed higher levels of CD49d and CD31.

Conclusion: These data indicate that PC at effector sites of the humoral response (BM and LP) show similar high differentiation, survival, and functional features but display a distinctive pattern of adhesion molecules, probably related to their respective homing locations.

Figure 1

Purification of plasma cells (PC) from human colon lamina propria (LP). The figure shows a representative example of the consecutive steps followed in the protocol used for the purification of LPPC. (A) Haematoxylin-eosin staining of a colon wall section showing the different layers. (B) Giemsa staining of the LP area showing abundant PC. (C, D) Haematoxylin-eosin staining of the dissected mucosal layer (including epithelium and LP areas) and the remaining tissue, respectively. (E) LP cells were obtained by collagenase digestion of the mucosal layer (C) and studied using labelled monoclonal antibodies (mAb) and flow cytometry analysis. A dot plot analysis of the CD19/CD38 cell expression is shown: a cell subset of CD38^h CD19^+/− can be observed. (F) Dot plot analysis of LP cells labelled for CD38 and CD54 showing the distinctive expression of CD54 by the LP CD38^h cells. (G) Dot plot analysis of CD38 and CD19 expression of LP CD54 selected cells showing the high degree of enrichment in CD38^h cells obtained by this procedure. (H, I) CD38^h cells purified by CD54 immunomagnetic selection were identified as PC by Giemsa staining as well as by intracytoplasmic IgA staining of cytospin preparations, respectively.

Figure 2

In vitro IgA secretion by lamina propria (LP) CD38^h cells. (A) Purified LP CD38^h cells were cultured for the indicated periods of time, and the quantity of IgA secreted into the supernatant was assessed by enzyme linked immunoabsorbent assay. Cycloheximide (Cx 10 μg/ml) was added to certain cultures. Results are expressed as μg/ml of IgA, and represent the mean (SEM) of four experiments. (B) LP CD38^h cells were cultured in the absence (Control) and presence (Ch11) of the anti-CD95 mAb CH11 (400 ng/ml) for seven days, and IgA secreted into the supernatant was determined. The results are expressed as the percentage of control IgA secretion, and represent the mean (SEM) of six experiments.

Figure 3

Differentiation and survival molecule expression by lamina propria (LP) and bone marrow (BM) plasma cells (PC). (A) Representative example (top series of histograms) of LPPC and BMPC (CD38^h cells) expression of several differentiation molecules (CD19, CD20, HLA-DR, and VS38c) and survival associated molecules (CD95, Bcl-2). Negative control histograms are shown in grey. (B) Mean (SEM) of several experiments (n≥5) for each marker, expressed as a percentage of positive PC. *p<0.05.

Figure 4

Messenger RNA for PRDI-BF1/B lymphocyte induced maturation protein 1 (Blimp 1) and B cell specific activation protein (BSAP) expression levels in different plasma cell (PC) populations. Relative amounts of Blimp-1 and BSAP mRNA were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) from highly purified tonsil PC (T-PC), lamina propria PC (LP-PC), and bone marrow PC (BM-PC). RT-PCR using β-actin mRNA sequences served as an internal standard. As a molecular weight marker, a Msp I digestion of pUC19 was used (MW). Data are representative of the results of three experiments using different donors.

Figure 5

Adhesion molecule expression by lamina propria (LP) and bone marrow (BM) plasma cells (PC). (A) Representative example (upper series of histograms) of LPPC and BMPC (CD38^h cells) expression of several adhesion molecules (CD21, CD44, α4β7, CD31, CD49d, and CD54). Negative control histograms are shown in grey. (B) Mean (SEM) of several experiments (n≥5) for each marker expressed as a percentage as well as mean fluorescence intensity (MFI) (C) of positive PC. *p<0.05.