Monocyte migration across the human blood-brain barrier

The potential contribution of circulating monocytes/macrophages to the inflammatory process of Alzheimer's disease (AD) is studied in a physiological model proposed by Fiala et al. [1].

Under normal circumstances, the blood-brain barrier (BBB) filters out the circulating monocytes from the brain tissue. However, chemotactic stimulus of inflammatory regions of the brain may attract such monocytes which then permeate the BBB. A large number of Aß deposits are present in the AD brain together with the presence of multiple inflammatory molecules. These are thought to induce the monocytes transmigration of the BBB, and movement into the vicinity of Aß deposits.

Differentiation of monocytes into macrophages:

• Human blood monocytes were obtained from peripheral blood of healthy donors.
• 1×10⁶ monocytes were incubated in 1 ml of RPMI medium during 8 days (alone or with different concentrations of Aß1-42).
• 400ml of RPMI and 20ml of MTT solution were added to each well, and incubated at 37 ^oC for 4 hrs.

• 1×10⁶ monocytes were cultured in 24-well plates using 1 ml of RPMI medium and various concentration of Aß1-42 for 48 hrs.

• TNF-a, IL-6, IL-10 and IL-12 levels in the culture media were obtained by a ßandwich" ELISA.
• IL-1b and MCP-1 (human monocyte chemoattractant protein-1) levels were obtained by using Quantikine ELISA.

• 160 mg of Aß1-42 in 35 ml of medium were placed in 6 wells, and 2×10⁴ monocytes in 1 ml of DME-S medium were added. After 16 hrs of incubation, 5×10⁵ monocytes in DME-S medium were added for an incubation time of 24 hrs.

Aß induces secretion of cytokines by cultured monocytes:

• 5×10⁵ monocytes were cultured in the presence of increasing concentrations of Aß to induce cytokine secretion. In figure 2 of Fiala's et al. we can observe different secretion levels for increasing concentrations of Aß. Concentrations were compared to that of the ones stimulated by the control media containing DMSO. The data obtained for the different Aß concentration levels:

Some Michaelis-Menten type functions are eye-fitted to the above data. If c is the cytokine concentration in pg/ml, and x is the Aß concentration. We have that for TNF-a secretion the function is

c =  cmaxx2  K+x2

where, c_max = 1400 and K = 2.5 (see figure 1).

Figure 1: Michaelis-Menten curve fitted to the data obtained from figure 2 A) of Fiala's et al. paper [1].

For IL-6 we found

c =  cmaxx2  K+x2

where, c_max = 140 and K = 2 (see figure 2).

Figure 2: Michaelis-Menten curve fitted to the data obtained from figure 2 B) of Fiala's et al. paper [1].

And for IL-12 we have

c =  cmaxx2  K+x2

where, c_max = 10.5 and K = 2 (see figure 3).

Figure 3: Michaelis-Menten curve fitted to the data obtained from figure 2 C) of Fiala's et al. paper [1].

There were some individual donor fluctuations in the amount of cytokine secretion when monocytes were stimulated with Aß1-42. Eight individual donors were investigated whose monocytes were treated with 2.5 µM of Aß1-42. In table 1 of Fiala's et al., we can compare the results. Again, concentrations were compared to those stimulated by the control media containing DMSO.

Aß1-42 with monocytes stimulate monocyte transmigrating across the BBB model:

The model consisted of placing a monolayer of human endothelial cells derived from cerebral microvessels and human astrocytes separating the vascular side (upper chamber) from the brain parenchymal side (lower chamber). In a control experiment, 5×10⁵ human monocytes were placed. Figure 4 of Fiala's et al. paper shows that:

• For only medium concentration, after 24 hrs of incubation time, around 700 monocytes migrated to the lower chamber ( ~ 1%).
• When 2×10⁴ monocytes were introduced into the lower chamber prior to the experiment, migration of monocytes from the upper to the lower chamber increased to approximately 3000 ( ~ 2%).
• Addition of 10 ml, 25 µM of Aß1-42 at the bottom of the lower chamber followed by medium resulted in the transmigration of 2×10⁴ monocytes in 24 hrs ( ~ 10%).
• When the lower chamber contained both, Aß1-42 and 2×10⁴ monocytes transmigration increased to 6.3×10⁴( ~ 26%).

It has been strongly suggested that inflammation plays a pivotal role in the pathogenesis of AD and development of dementia. In vitro cultures of human microglia obtained from rapid autopsies from individuals with AD secreted 100-fold or more IL-1b, IL-6 and TNF-a than those cells from nondemented subjects. In this study fluctuations in cytokine secretion elicited by Aß in different donors are thought to be due to the individual's immunological responsiveness. Also, in this study the authors show the proinflammatory effects of Aß1-42 on peripheral monocytes. It is suggested that some of the microglia-like cells surrounding the Aß fibrils are transmigrated monocytes from the BBB. In a dose and time-dependent manner, it was shown how Aß induced differentiation of monocytes into macrophages, as well as some monocyte migration across the BBB.
 

Reference:

[1] Fiala M, Zhang L, Gan X, Sherry B, Taub D, Graves MC, Hama, S, Way D, Weinand M, Witte M, Lorton D, Kuo Y_M, Roher AE (1998). Amyloid-beta induces chemokine secretion and monocyte migration across human blood-brain barrier model. Molec Med 4: 480-489.Abstract