Osteopontin prevents monocyte recirculation and apoptosis

Abstract

Cells of the monocyte/macrophage lineage have been shown to be the principal targets for productive HIV-1 replication within the CNS. In addition, HIV-1-associated dementia (HAD) has been shown to correlate with macrophage abundance in the brain. Although increased entry of monocytes into the brain is thought to initiate this process, mechanisms that prevent macrophage egress from the brain and means that prevent macrophage death may also contribute to cell accumulation. We hypothesized that osteopontin (OPN) was involved in the accumulation of macrophages in the brain in neuroAIDS. Using in vitro model systems, we have demonstrated the role of OPN in two distinct aspects of macrophage accumulation: prevention from recirculation and protection from apoptosis. In these unique mechanisms, OPN would aid in macrophage survival and accumulation in the brain, the pathological substrate of HAD.

Figure 1. OPN is not a classic chemokine for monocytes

Control is shown in slashed boxes, OPN in grey, CX3CL1 (fractalkine) in black and CCL2 in white. Values are relative to control and shown is one representative experiment out of three performed. (A) Shown are the CD14+CD16+ monocyte migration indices. CX3CL1 is known to be a potent chemoattractant for CD14+CD16+ monocytes. All OPN indices shown are similar to control. (B) Shown are the CD14+CD16- monocyte migration indices. CCL2 is known to be a potent chemoattractant for CD14+CD16- monocytes. All OPN indices shown are similar to control. 1, 5, and 20 nM of OPN corresponds to concentrations of 65, 325, and 1300 ng/ml; 1, 5, and 20 nM of CX3CL1 corresponds to concentrations of 90, 450, and 1800 ng/ml; 1, 4, and 20 nM of CCL2 corresponds to concentrations of 8.7, 35, and 87 ng/ml.

Figure 2. In vitro model for the examination of the reverse transmigration of human monocytes

(A) Diagram of the in vitro filter model to study reverse transmigration. (B & C) Tight junctions between adjacent HUVECs; calibration bars: 100 nm & 200 nm respectively (Individual insets show enlargements of the junctions). (D - F) Three figures illustrating monocytes in transit through the layer of endothelial cells (*) into the collagen matrix; calibration bars: 2 μm, 2 μm & 1 μm respectively.

Figure 3. Osteopontin decreased the percent of monocytes that reverse transmigrated

(A) Osteopontin (750 ng/ml) significantly decreased the percent of total monocytes that reverse transmigrated compared to control (Student’s t test, p=0.02). (B) There is a decrease (however, non-significant p>0.05) in the percent of CD14+CD16- monocytes that reverse transmigrated compared to control. (C) A significant decrease in the amount of CD14+CD16+ monocytes that reverse transmigrated was demonstrated between OPN and control (Student’s t test p=0.01). The mean of 3 experiments with standard error of the mean is shown.

Figure 4. Osteopontin protects monocytes from apoptosis

(A) In eight separate experiments (EXP 1-8), human monocytes were grown in non-adherent culture conditions for 24 hours. Shown is the percent of living cells (Annexin V and PI neg. cell population) in RPMI or cultured with 750 ng/ml of osteopontin (OPN). The fold is the increase in OPN compared to RPMI only. (B) The averages and standard error of the means are shown. OPN significantly increased the percentage of viable monocytes (4.7-fold, p= 0.0005).

Figure 5. FACS staining showed OPN decreased the percent of monocyte apoptosis

Annexin V-FITC and PI staining of two representative patients are shown (A and B). The left panel shows the cells cultured in RPMI for 24 hrs, and the right panel shows cells that were cultured in RPMI with 750 ng/ml of OPN for 24 hrs. Note that OPN increases live cell percentages on the lower left quadrant compared to control.

Figure 6. OPN increases CD44v6 and integrin expression

Arrows indicate the histograms for cells cultured in RPMI media (grey line) and OPN (black line). (A) CD44v6 expression in monocytes that were gated for Annexin V neg and PI neg (alive) cells after 24 hrs of culture is shown in three patients. (B) Integrin αVβ3 in monocytes that were gated for Annexin V neg and PI neg (alive) cells after 24 hrs of culture is shown in three patients.