Relevance of distinct monocyte subsets to clinical course of ischemic stroke patients

Abstract

Background and purpose: The most common strategy for treating patients with acute ischemic stroke is thrombolytic therapy, though only a few patients receive benefits because of the narrow time window. Inflammation occurring in the central nervous system (CNS) in association with ischemia is caused by immune cells including monocytes and involved in lesion expansion. If the specific roles of monocyte subsets in stroke can be revealed, they may become an effective target for new treatment strategies.

Methods: We performed immunological examinations of 36 consecutive ischemic stroke patients within 2 days of onset and compared the results with 24 age-matched patients with degenerative disorders. The stroke patients were repeatedly tested for the proportions of monocyte subsets in blood, and serum levels of pro- and anti-inflammatory cytokines immediately after admission, on days 3-7 and 12-16 after stroke onset, and on the day of discharge. In addition, immunological measurements were analyzed for relationships to stroke subtypes and complications, including progressive infarction (PI) and stroke-associated infection (SAI).

Results: Monocyte count was significantly increased from 0-16 days after stroke as compared to the controls (p<0.05). CD14(high)CD16(-) classical and CD14(high)CD16(+) intermediate monocytes were significantly increased from 0-7 and 3-16 days after stroke, respectively (p<0.05), whereas CD14 (dim)CD16(high) non-classical monocytes were decreased from 0-7 days (p<0.05). Cardioembolic infarction was associated with a persistent increase in intermediate monocytes. Furthermore, intermediate monocytes were significantly increased in patients with PI (p<0.05), while non-classical monocytes were decreased in those with SAI (p<0.05). IL-17A levels were positively correlated with monocyte count (r=0.485, p=0.012) as well as the percentage of non-classical monocytes (r=0.423, p=0.028), and negatively with that of classical monocytes (r=-0.51, p=0.007) during days 12-16.

Conclusions: Our findings suggest that CD14(high)CD16(+) intermediate monocytes have a role in CNS tissue damage during acute and subacute phases in ischemic stroke especially in relation to cardioembolism.

Figure 1. Analysis of monocyte subsets.

(A) Monocytes were gated in a circle using forward scatter (FSC) and side scatter (SSC) plots. (B) Classical monocytes were identified by high expression of CD14 and no expression of CD16 (CD14^highCD16^-), intermediate monocytes by high expression of CD14 and various levels of positivity for the CD16 molecule (CD14^highCD16⁺), and non-classical monocytes by scant expression of CD14 and high expression of CD16 (CD14 ^dimCD16^high). The proportions of each subset were evaluated by comparing the number of cells (dots) in the individual compartment to the total number of monocytes enclosed in the circle designated as P1.

Figure 2. Time courses of monocyte subsets after stroke.

(A) The overall number of circulating monocytes was increased. The percentages of (B) classical and (C) intermediate Mo were increased, whereas that of (D) non-classical Mo was decreased. Bars show the mean ± SEM. *p<0.05, t-test, compared to control. Control, n=24; day 0-2, n=36; day 3-7, n=35; day 12-16; n=33; day of hospital discharge, n=26.

Figure 3. Correlation between monocytes and NIHSS score on admission.

(A) There was a significant positive correlation between overall monocyte count and NIHSS score, (B–D) while none was seen between the monocyte subsets and that score.

Figure 4. Stroke subtype and monocyte subsets from day 0-2 (hyperacute phase).

(A) The overall monocyte counts in the LAA and CE groups were significantly increased as compared to the control. (B, C, D) The percentages for the monocyte subsets in the CE group were significantly changed as compared to the control. Bars show the mean ± SEM. *p<0.05, t-test, compared to control. †p<0.05, ANOVA, compared to CE. C: control; LAA: large artery atherosclerosis; CE: cardioembolism; SAO: small artery occlusion.

Figure 5. Stroke subtypes and intermediate monocytes after hyperacute phase of stroke.

(A–C) A significantly elevated percentage of intermediate Mo in the CE group persisted until the day of discharge. *p<0.05, t-test, as compared to control. C: control; LAA: large artery atherosclerosis; CE: cardioembolism; SAO: small artery occlusion.

Figure 6. Time courses of monocyte subsets regarding stroke complications.

Classical and intermediate Mo were increased, while non-classical Mo were decreased. Patients were divided into (A) with or without PI, (B) excluding SAI, (C) and with or without SAI. *p<0.05, t test, compared to control. †p<0.05, t test, compared to other group in category. PI = progressing infarction, SAI = stroke-associated infection.

Figure 7. Dynamics of pro-inflammatory cytokines after stroke.

(A) In the SAI group (closed diamond), serum IL-6 levels were elevated from 0–7 days after stroke, (B) while serum IL-17A levels were elevated in the PI group without SAI (closed square) from 3–7 days after stroke.

Figure 8. Relationship between monocyte subsets and serum IL-17A in early subacute phase (12-16 days) after stroke.

IL-17A level was positively correlated with (A) monocyte count and (D) percentage of non-classical Mo, and negatively with (B) percentage of classical Mo. Statistical analysis was performed using Spearman’s rank correlation coefficient.