Application of immunoaffinity capillary electrophoresis to the measurements of secreted cytokines by cultured astrocytes

Abstract

The ability of the central nervous system (CNS) to act in conjunction with the immune system has been of great interest to both neurobiologists and immunologists. Previous studies have shown that astrocytes can be stimulated, by various peptides, to act as immune regulators within the CNS and release cytokines and chemokines. However, the regulatory mechanism of astrocytes is still poorly understood. Our present study describes a micro-device capable of monitoring the growth and stimulation of 20 astrocytes by vasoactive intestinal peptide. A microdialysis needle was used to collect the secretion by products, which were analyzed by immunoaffinity capillary electrophoresis (ICE) for the secretion of pro-inflammatory cytokines, IL-1alpha, IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha; hemopoietic cytokines, IL-3, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF); and chemokines; regulated upon activation normal T-cell expression sequence (RANTES), macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. Vasoactive intestinal peptide stimulated astrocytes showed an almost immediate release of pro-inflammatory cytokines and chemokines, with an increase over baseline ranging from 3 to 15 fold, while no substantial increase over baseline was observed for hemopoietic cytokines. This system demonstrates the ability to isolate individual cells in a closely controlled environment and identify and quantify their analytes.

Figure 1

Drawing of A) top view and B) side view of the single cell micro-incubation chamber with the microdialysis sampling probe cemented in place.

Figure 2

Illustration of the preparation of an immunoaffinity capillary. Step 1: The antibody is digested by pepsin digestion, yielding the F(Ab)₂′ fragment, which is further reduced by Clelands’ reagent to 2 FAb fragments. Step 2: The antibody fragments are loaded by capillary action into the derivatized capillary. Step 3: The AlexaFluor labeled analytes are introduced into the capillary and bind to the immobilized antibody. Nonreactive analytes are removed. Step 4: An elution buffer is introduced into the capillary, breaking the antibody/analyte bond and releasing the analytes, which separate and are detected as an electropherogram.

Figure 3

An electropherogram of the 11 cytokine/chemokine standards run on the ICE system. The IL-11 marker is not shown in the electropherograms.

Figure 4

Electropherograms of cytokine/chemokine secretions from VIP stimulated astrocytes. While samples were collected every 5 min, electropherograms are depicted every 10 min for clarity. Peaks from left to right are: M-CSF, GM-CSF, G-CSF, IL-6, IL-3, TNF-α, IL-1α, IL-1β, MIP-1α, MIP-1β, and RANTES. The IL-11 marker is not shown in the electropherograms.

Figure 5

Graph of time (min) versus amount of analytes recovered (pg/mL) for pro-inflammatory cytokines recovered from VIP stimulated astrocytes.

Figure 6

Graph of time (min) versus amount of analytes recovered (pg/mL) for hemopoetic cytokines recovered from VIP stimulated astrocytes.

Figure 7

Graph of time (min) versus amount of analytes recovered (pg/mL) for chemokines recovered from VIP stimulated astrocytes.