CD45RA and CD45RO Are Regulated in a Cell-Type Specific Manner in Inflammation and Sepsis

Abstract

CD45 is a transmembrane glycoprotein that is located on the surface of all leukocytes and modulates both innate and adaptive immune system functions. In a recent study, inflammation modulated the CD45 expression in leukocytes, but the effect on the expression of CD45 subtypes is unknown. In the present study, we therefore investigated the effect of inflammatory conditions in humans (surgery, sepsis) and ex vivo incubation with lipopolysaccharides (LPS) on the expression of the subtypes CD45RA and CD45RO in granulocytes, lymphocytes, and monocytes. Whole blood samples were obtained from healthy volunteers, postoperative patients, and patients with sepsis at day 1 of diagnosis, respectively. Samples were incubated with fluorescent antibodies directed against CD45, CD45RA and CD45RO in the absence and presence of lipopolysaccharide and subjected to flow cytometry. In comparison to volunteers, CD45RA surface expression in postoperative and septic patients was reduced by 89% exclusively on granulocytes, but not on lymphocytes or monocytes. In contrast, CD45RO was exclusively reduced on lymphocytes, by 82%, but not on other cell types. Receiver operating characteristic curve analyses demonstrated that CD45RA (on granulocytes) and CD45RO (on lymphocytes) allow a good differentiation of volunteers and patients with sepsis (AUC = 0.9; p = 0.0001). The addition of LPS to the whole blood samples obtained from volunteers, postoperative patients, and septic patients markedly increased the CD45RO expression in granulocytes, lymphocytes, and monocytes. In contrast, LPS reduced CD45RA exclusively on monocytes. In conclusion, the surface expression of CD45RA and CD45RO is regulated in inflammation in a cell-type- and stimulus-specific manner. Considering that CD45 subtypes are critically involved in immune system signaling, the pathophysiologic and diagnostic implications warrant further investigation.

Keywords: CD45; CD45RA; CD45RO; granulocytes; inflammation; lipopolysaccharides; lymphocytes; monocytes; sepsis.

Figure 1

Diagrams (A,B) demonstrate the gating of granulocytes, lymphocytes, and monocytes. Diagram (C) shows the surface expression of CD45, CD45RA, and CD45RO in healthy volunteers. For the gating sideward scatter area (SSC-A), CD45 and CD14 were used.

Figure 2

Representative flow cytometry diagrams demonstrating the expression of CD45RA in granulocytes and the expression of CD45RO in lymphocytes in volunteers, postoperative patients, and patients with sepsis. Expression of both subtypes was markedly reduced in patients with sepsis.

Figure 3

Granulocyte CD45, CD45RA, and CD45RO expression in volunteers, postoperative patients, and patients with sepsis in absence of LPS (upper lane) and presence of LPS (lower lane). The mean fluorescence intensity was determined using flow cytometry of antibody treated cells. Results are shown as individual data points as well as mean and standard error of the mean. p-values denote Bonferroni corrected t-test results as the post hoc test performed subsequent to a significant ANOVA result.

Figure 4

Lymphocyte CD45, CD45RA, and CD45RO in volunteers, postoperative patients, and patients with sepsis in absence of LPS (upper lane) and presence of LPS (lower lane) Cells were labeled with antibodies and fluorescence intensity was measured using flow cytometry. Results are shown as individual data points as well as mean fluorescence intensity and standard error of the mean. p-values denote Bonferroni corrected t-test results as the post hoc test performed subsequent to a significant ANOVA result.

Figure 5

Monocyte CD45, CD45RA, and CD45RO in volunteers, postoperative patients, and patients with sepsis in absence of LPS (upper lane) and presence of LPS (lower lane). Whole blood samples were incubated with antibodies and subjected to flow cytometry. Data of the mean fluorescence intensity are shown as individual data points as well as mean and standard error of the mean. p-values denote Bonferroni corrected t-test results as the post hoc test performed subsequent to a significant ANOVA result.

Figure 6

Receiver operating characteristic curves, area under the curve, and asymptomatic significance levels demonstrating the capability of CD45RA in granulocytes and CD45RO in lymphocytes to differentiate between volunteers and patients with sepsis. Addition of LPS did not affect the excellent discrimination between volunteers and patients with sepsis.

Figure 7

Distribution of granulocytes, lymphocytes, and monocytes in volunteers, postoperative patients, and patients with sepsis. Data are given as the percentage of the respective leucocyte subtype to all subtypes (100%) as gated with flow cytometry. p-values denote Bonferroni corrected t-test results as the post hoc test performed subsequent to a significant ANOVA result.