Human umbilical cord blood cell therapy blocks the morphological change and recruitment of CD11b-expressing, isolectin-binding proinflammatory cells after middle cerebral artery occlusion

Abstract

Secondary neurodegeneration resulting from stroke is mediated by delayed proinflammatory signaling and immune cell activation. Although it remains unknown which cell surface markers signify a proinflammatory phenotype, increased isolectin binding occurs on CD11b-expressing immune cells within injured brain tissue. Several reports have confirmed the efficacy of human umbilical cord blood (HUCB) cell therapy in reducing ischemic injury in rat after middle cerebral artery occlusion (MCAO), and these effects were attributed in part to dampened neuroinflammation. The present study examined the time course of lectin binding to cells of microglia/macrophage lineage within 96 hr after MCAO and whether delayed HUCB cell treatment alters the migration and/or morphological characteristics of these cells throughout the period of infarct expansion. Isolectin binding was up-regulated in response to injury, was maximal at 96 hr, and colocalized with cells that expressed the putative proinflammatory markers MMP-9 and nitric oxide. Isolectin-tagged fluorescence was also significantly increased at 72 hr and localized to greater numbers of amoeboid, CD11b-expressing cells relative to 51 hr. Treatment with 1 x 10(6) HUCB cells significantly reduced total lectin binding at 72 hr, as well as the total area occupied by lectin-tagged fluorescence at both 51 and 72 hr, relative to vehicle-treated controls. This effect was accompanied by a shift in the morphology of CD11b-positive cells from amoeboid to ramified shape. These data indicate that HUCB cell therapy suppressed the recruitment of proinflammatory, isolectin-binding cells during the period of infarct expansion, thus offering a potential mechanism for the protective effects of HUCB cell therapy.

Figure 1. CD11b and isolectin colocalize within the striatal infarct at delayed time points

CD11b-positive cells in sham-MCAO animals displayed a ramified shape (A) and did not bind isolectin, which only labeled blood vessels (B). Inset (C) shows Fluoro-Jade staining within the striatal infarct at 48 hrs. CD11b immunoreactivity was localized to many hypertrophic ramified cells (C) that also bound isolectin (D) 48 hrs after MCAO. At 96 hrs, colocalization of CD11b (E) and isolectin (F) was abundant in cells that displayed amoeboid morphology. Scale bars = 50 μm.

Figure 2. Quantification of isolectin binding in the infarcted striatum after MCAO

Isolectin-tagged fluorescence was quantified at 3, 24, 48 and 96 hrs after MCAO (n=3 rats per group, 3 sections per rat). Mean values were normalized to counts obtained from sham-operated rats. Total fluorescent signal did not significantly differ between the 3, 24 and 48 hr timepoints. Isolectin binding was significantly increased at 96 hrs compared to both 3 and 48 hrs (* p<0.02), while a trend toward an increase was observed at 24 hrs (p=0.06).

Figure 3. Isolectin-binding cells express neuroinflammatory markers in vivo and ex-vivo

MMP-9 expression was ubiquitous throughout the striatal infarct 96 hrs after MCAO (A). Isolectin binding was detected on microglia/macrophages throughout the infarct (B) and colocalized with MMP-9-expressing cells (C). Isolectin binding was also prominent in organotypic hippocampal slices exposed to OGD (D), and DAF-FM staining (E) showed that NO production occurred in isolectin-binding cells (F). Scale bars = 100 μm.

Figure 4. Isolectin-binding immune cells migrate into the striatal infarct at 51 hrs

Rats that received vehicle at 48 hrs post-MCAO showed a robust immune cell response at 51 hrs. CD11b-immunoreactivity was present on cells within the corpus striatum (A) and peri-infarct regions adjacent to the corpus callosum (B) and lateral ventricle (C). Isolectin-binding cells were also present in the striatum (D) and displayed amoeboid morphology consistent with that of CD11b-expressing cells (A,D, insets). Isolectin-tagged fluorescent cells were present in greater quantities both in and adjacent to the corpus callosum (E) as well as in the periventricular region (F), where they were densely distributed between blood vessels originating at the base of the brain (F, inset) and peri-infarct striatal tissue. Scale bars: A,D insets = 50 μm; all others = 100 μm. CC = corpus callosum. LV = lateral ventricle.

Figure 5. CD11b-expressing and Isolectin-binding immune cells invade the striatal infarct at 72 hrs

Rats that received vehicle 48 hrs post-MCAO exhibited a robust neuroinflammatory response at 72 hrs. Cells displaying CD11b immunoreactivity (A) and isolectin-tagged fluorescence (D) infiltrated the striatal infarct in greater quantities relative to the 51 hr timepoint. Isolectin-binding colocalized to many CD11b-positive, amoeboid shaped immune cells (B,E). Both CD11b-expressing (C) and isolectin-binding (F) cells were also greatly elevated in peri-infarct tissues adjacent to the corpus callosum. Scale bars: A,C,D,F = 100 μm; B,E = 50 μm. CC = corpus callosum.

Figure 6. Delayed HUCB cell treatment blocks immune cell migration to the striatal infarct at 51 hrs

Rats that received HUCB cell treatment 48 hrs post-MCAO showed marked reductions in immune cell migration to the striatal infarct. CD11b immunoreactivity was localized to immune cells within the striatal infarct (A) where lectin-binding cells were also found (D). These cells displayed hypertrophic ramified and amoeboid morphologies (A,D, insets) similar to vehicle-treated animals, but were far fewer in number. HUCB cell therapy greatly reduced immune cell infiltration into the periventricular region (B,E), and the few CD11b/lectin-positive cells present displayed a ramified morphology (C,F, arrows) that resembled microglia/macrophages in sham-MCAO rats. Scale bars = A,D insets, C,F = 50 μm; all others = 100 μm. LV = lateral ventricle.

Figure 7. Delayed HUCB cell treatment blocks the infiltration of CD11b- and lectin-positive cells at 72 hrs

Rats that received HUCB cell treatment 48 hrs post-MCAO showed a sparse distribution of CD11b-immunopositive microglia/macrophages within the striatum (A), and these cells displayed ramified morphology (B) characteristic of the inactivated state. Isolectin-tagged fluorescence was detected on blood vessels throughout the striatum (D) but did not label CD11b-expressing cells (E). Peri-infarct regions both in and adjacent to the corpus callosum showed reductions in the numbers of CD11b immunoreactive cells (C), and were devoid of lectin-binding cells (F). Scale bars: A,C,D,F = 100 μm; B,E = 50 μm. CC = corpus callosum.

Figure 8. Quantification of isolectin binding

Total isolectin-tagged fluorescent signal (A) and total area occupied by isolectin-positive cells (B) were quantified in tissues from rats treated with HUCB cells or vehicle at 48 hrs post-MCAO and sacrificed at 51 or 72 hrs. Isolectin binding was significantly elevated in vehicle-treated animals at 72 hrs relative to the 51 hr timepoint, and treatment with HUCB cells significantly reduced isolectin binding at 72 hrs relative to vehicle-treated rats at both timepoints. Treatment with HUCB cells also reduced isolectin-positive area at both timepoints relative to vehicle-treated controls. Asterisk denotes significance from 51 hr vehicle (p<0.01). Pound sign denotes significance from 72 hr vehicle (p<0.01).