Dopamine Increases CD14+CD16+ Monocyte Transmigration across the Blood Brain Barrier: Implications for Substance Abuse and HIV Neuropathogenesis

Abstract

In human immunodeficiency virus-1 (HIV) infected individuals, substance abuse may accelerate the development and/or increase the severity of HIV associated neurocognitive disorders (HAND). It is proposed that CD14⁺CD16⁺ monocytes mediate HIV entry into the central nervous system (CNS) and that uninfected and infected CD14⁺CD16⁺ monocyte transmigration across the blood brain barrier (BBB) contributes to the establishment and propagation of CNS HIV viral reservoirs and chronic neuroinflammation, important factors in the development of HAND. The effects of substance abuse on the frequency of CD14⁺CD16⁺ monocytes in the peripheral circulation and on the entry of these cells into the CNS during HIV neuropathogenesis are not known. PBMC from HIV infected individuals were analyzed by flow cytometry and we demonstrate that the frequency of peripheral blood CD14⁺CD16⁺ monocytes in HIV infected substance abusers is increased when compared to those without active substance use. Since drug use elevates extracellular dopamine concentrations in the CNS, we examined the effects of dopamine on CD14⁺CD16⁺ monocyte transmigration across our in vitro model of the human BBB. The transmigration of this monocyte subpopulation is increased by dopamine and the dopamine receptor agonist, SKF 38393, implicating D1-like dopamine receptors in the increase in transmigration elicited by this neurotransmitter. Thus, elevated extracellular CNS dopamine may be a novel common mechanism by which active substance use increases uninfected and HIV infected CD14⁺CD16⁺ monocyte transmigration across the BBB. The influx of these cells into the CNS may increase viral seeding and neuroinflammation, contributing to the development of HIV associated neurocognitive impairments.

Keywords: CD14+CD16+ monocytes; CNS HIV reservoirs; Dopamine; HAND; Neuroinflammation; Substance abuse.

Fig. 1

The frequency of CD16⁺ monocytes in the peripheral blood of HIV infected individuals is increased with active substance use and dopamine increases the transmigration of CD14⁺CD16⁺ monocytes across the BBB. a PBMC from HIV seronegative donors (n=12) and from HIV seropositive donors from the MHBB cohort (n=48) were stained with fluorochrome-coupled CD14, CD16 or isotype matched negative control antibodies and analyzed by flow cytometry. The total number of CD14⁺ monocytes was determined and the percentage of these monocytes that were CD14⁺CD16⁺ and CD14^lowCD16⁺ was calculated. HIV infected donors were divided into non-active drug users (n=22) and active drug users (n=26). Data points for each donor are shown with mean ± SD. Significance was determined using two-tailed unpaired Mann-Whitney test. **p<0.001; ***p<0.0001. b PBMC from HIV seropositive donors from the WIHS cohort were added to BBB cocultures and CD14⁺CD16⁺ monocyte and CD3⁺ T cell transmigration in response to 10 μM dopamine (DA) was expressed as fold increase over baseline (media plus dH₂O), which was set to one (dotted line) for each independent donor. Data are expressed as mean ± SEM. Significance was determined by two-tailed Wilcoxon signed-rank test comparing medians for each treatment group to a hypothetical value of one. *p<0.05 (DA 10 μM compared to baseline); n=9 each.

Fig. 2

Cultured CD14⁺CD16⁺ monocytes, but not CD3⁺ T cells, transmigrate across the BBB in response to dopamine. a Transmigration experiments were performed with mature monocyte cultures derived from HIV seronegative leukopaks. CD14⁺CD16⁺ monocyte transmigration across the BBB in response to dopamine (DA) and/or CXCL12 was expressed as fold increase over baseline (media plus 0.1% BSA in PBS), which was set to one (dotted line) for each independent donor. Data are expressed as mean ± SEM. Significance was determined by two-tailed Wilcoxon signed-rank test comparing medians for each treatment group to a hypothetical value of one. ***p<0.001 (DA 10 μM compared to baseline, n=15; DA 20 μM compared to baseline, n=13; CXCL12 25 ng/ml compared to baseline, n=14). Transmigration in response to CXCL12+DA was not significant compared to DA alone. b Transmigration experiments performed with PBMC from HIV seronegative leukopaks to quantify CD3⁺ T cell transmigration. Data are expressed as mean ± SEM. Significance was determined by two-tailed Wilcoxon signed-rank test comparing medians for each treatment group to a hypothetical value of one. *p<0.05 (CXCL12 25 and 50 ng/ml compared to baseline); n=6 each.

Fig. 3

Dopamine increases cell membrane protrusions in CD14⁺CD16⁺ monocytes. a Cells from a mature monocyte culture were treated with media plus dH₂O (Untreated) or 20 μM dopamine (DA) for 15 min and stained for actin (Texas Red®-X phalloidin) (red staining), tubulin (FITC) (green staining) and nuclei (DAPI) (blue staining). Cell membrane protrusions with colocalized actin and tubulin staining, indicative of a chemotactic phenotype and indicated by the arrows, were identified by confocal microscopy. b The percentage of mature monocytes with membrane protrusions in untreated and dopamine (DA) treated cultures was quantified. The data were expressed as fold change compared to untreated cells, which was set to one for each independent donor as indicated by the dotted line. Data are expressed as mean ± SD. Significance was determined by two-tailed Wilcoxon signed-rank test comparing medians for each treatment group to a hypothetical value of one. *p<0.05 (DA 20 μM compared to untreated); n=6.

Fig. 4

Dopamine does not increase cell membrane protrusions in T cells. a Purified T cells from one representative donor treated with media plus 0.1% BSA (Untreated), 20 μM dopamine (DA), or 50 ng/ml CXCL12 for 15 min were fixed and stained for actin (Texas Red®-X phalloidin), tubulin (FITC) and nuclei (DAPI). Membrane protrusions were detected by colocalized actin and tubulin staining as indicated by the arrows. b T cells were treated with 20 μM dopamine (DA) or 50 ng/ml CXCL12 for 15, 30 or 45 min. The percentage of T cells with membrane protrusions was quantified at the time point of optimal membrane protrusion formation with CXCL12 in each experiment. The data were then expressed as fold change compared to untreated cells, which was set to one for each independent donor as indicated by the dotted line. Data are expressed as mean ± SD. Significance was determined by two-tailed Wilcoxon signed-rank test comparing medians for each treatment group to a hypothetical value of one. *p<0.05 (CXCL12 50 ng/ml compared to untreated); n=6.

Fig. 5

Dopamine increases active ADAM17 in CD14⁺CD16⁺ monocytes, but not in T cells. Mature monocytes and T cells from the same leukopak were treated with media plus dH₂O (Un), 10 μM dopamine (DA), or 100 ng/ml PMA for 5 min. Cell lysates prepared from these cells were then analyzed by Western blot for active ADAM17 and β-actin.

Fig. 6

CD14⁺CD16⁺ monocytes express greater surface D1R and D5R, and less D4R, than T cells. Mature monocytes and T cells from independent donors were incubated with antibodies to D1R, D5R, D4R or negative control rabbit serum or goat IgG. After incubation in primary antibody, cells were incubated with PE-labeled anti-rabbit or anti-goat IgG secondary antibody. Stained cells were analyzed by flow cytometry and the MFI was calculated by subtracting the appropriate negative control antibody or serum staining from the staining obtained with each dopamine receptor antibody. Significance was determined by two-tailed unpaired Mann-Whitney test. **p<0.01 (D5R on mature monocytes, n=17, compared to T cells, n=9); ***p<0.001 (D1R on mature monocytes, n=19, compared to T cells, n=10) (D4R on mature monocytes, n=18, compared to T cells, n=9).

Fig. 7

CD14⁺CD16⁺ monocytes transmigrate across the BBB in response to the D1-like dopamine receptor agonist, SKF38393. Quantification of CD14⁺CD16⁺ monocyte transmigration across the BBB in response to different concentrations of SKF38393 or dopamine (DA) expressed as fold increase over baseline (media plus dH₂O), which was set to one as indicated by the dotted line. Data are expressed as mean ± SEM. Significance was determined by two-tailed Wilcoxon signed-rank test comparing medians for each treatment group to a hypothetical value of one. *p<0.05 (SKF38393 1 μM and 50 μM compared to baseline, n=6 each) (DA 1 μM compared to baseline, n=11); **p<0.01 (SKF38393 10 μM and DA 50 μM compared to baseline, n=8 each) (DA 10 μM compared to baseline, n=10).

Fig. 8

Schematic representation of the proposed mechanism by which dopamine increases chemokine induced CD14⁺CD16⁺ monocyte transmigration across the BBB in HIV infected substance abusers. In areas of the CNS with dopaminergic neuronal synapses in close proximity to BBB microvasculature, active substance use will increase the concentration of dopamine, resulting in the activation D1R/D5R receptors on the leading edge of CD14⁺CD16⁺ monocytes transmigrating across the BBB in response to chemokines, including CCL2. Upon dopamine receptor activation, ADAM 17 on the cell surface of the lagging edge of the monocyte is activated, resulting in cleavage of monocyte Mac-1 bound to endothelial ICAM-1 and the accelerated completion of transmigration.