Preferential production of interferon-gamma by CD4+ T cells expressing the homing receptor integrin alpha4/beta7

Abstract

Recent studies indicate that T helper type 1 (Th1) and 2 (Th2) lymphocytes differ in their expression of molecules that control T-cell migration, including adhesion molecules and chemokine receptors. We investigated the relationship between cytokine production and expression of the homing receptor integrin alpha4/beta7 on T cells. We began by analysing cytokine production by human CD4+ CD45RA- memory/effector T cells following brief (4 hr) stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. alpha4/ beta7high CD4+ T cells were more likely to produce the Th1 cytokine interferon-gamma (IFN-gamma) than were alpha4/beta7- CD4+ T cells in all six subjects studied. In contrast, production of the Th2 cytokine interleukin-4 (IL-4) was similar on alpha4/ beta7high and alpha4/beta7- CD4+ T cells. In addition, we found that human CD4+ CD45RA- T cells that adhered to the alpha4/beta7 ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) had a greater capacity to produce IFN-gamma than did non-adherent cells, suggesting that the association between alpha4/beta7 expression and IFN-gamma production has functional significance. These results suggested that primary activation under Th1-promoting conditions might favour expression of alpha4/beta7. We directly examined this possibility, and found that naïve murine CD4+ T cells activated under Th1-promoting conditions expressed higher levels of alpha4/beta7 compared to cells activated under Th2-promoting conditions. The association between alpha4/beta7 expression and IFN-gamma production by CD4+ T cells may help to determine the cytokine balance when MAdCAM-1 is expressed at sites of inflammation in the intestine or elsewhere.

Figure 1

Association between β₇ expression and IFN-γ production by human CD4⁺ CD45RA^– memory/effector T cells. Human PBMC were isolated and cultured for 4 hr without a stimulus (a–d), or in the presence of PMA and ionomycin (e–h). Expression of integrin β₇ and CD45RA on unstimulated (a) and stimulated (e) CD4⁺ T cells. CD4⁺ T cells were divided into CD45RA⁺ (naïve) and CD45RA^– (memory/effector) and β₇^– subsets, as shown. IFN-γ production was then determined for , β₇^– and CD45RA⁺ CD4⁺ T cells for the unstimulated (b–d) and stimulated (f–h) samples.

Figure 2

Cytokine production by and β₇^– human blood CD4⁺ CD45RA^– T cells from six normal subjects. Production of the cytokines IFN-γ (a), TNF-α (b), IL-2 (c), and IL-4 (d) were measured by intracellular cytokine staining after 4 hr stimulation with PMA and ionomycin. The association between cytokine production and β₇ expression on CD4⁺ CD45RA^– T cells was determined using the approach outlined in Fig. 1.

Figure 3

Cytokine production by and β₇^– human blood CD8⁺ CD45RA^– T cells from six normal subjects. The experimental protocol and analysis were performed in the same manner as described in Figs 1 and 2, except that a CD8 antibody was used for staining cells (instead of a CD4 antibody). Columns labelled ‘0’ indicate that no cytokine production was detected for these subsets.

Figure 4

Preferential production of IFN-γ by MAdCAM-1-adherent CD4⁺ memory/effector T cells. Human PBL were allowed to adhere to MAdCAM-1- or ICAM-1-coated plates, and adherent and non-adherent populations were treated with PMA and ionomycin for 4 hr to stimulate cytokine production. IFN-γ production by CD4⁺ CD45RA^– T cells was analysed by intracellular cytokine staining of (a) MAdCAM-1-adherent, (b) MAdCAM-1-non-adherent, (c) ICAM-1-adherent and (d) ICAM-1-non-adherent populations. The expression of β₇ on CD4⁺ CD45RA^– T cells in these populations was also analysed by flow cytometry (e–h).

Figure 5

Association between β₇ expression and IFN-γ production by mouse CD4⁺ T cells activated and polarized in vitro. CD4⁺ T cells from DO11.10 T-cell receptor-transgenic mice were activated using anti-CD3 plus anti-CD28 in the presence of IL-2 alone (a), or in combination with exogenous IL-12 plus anti-IL-4 antibody (Th1 conditions) (b) or exogenous IL-4 plus anti-IFN-γ (Th2 conditions) (c). After 12 days, cells were re-stimulated with PMA and ionomycin for 4 hr and β₇ integrin expression and IFN-γ production were analysed by flow cytometry (see Materials and Methods). The percentage of cells in each quadrant is shown at the upper right corner of each plot. Quadrant positions were established based on staining with isotype control antibodies (99% of cells were in the lower left quadrant when control antibodies were used for staining, not shown).

Figure 6

α₄/β₇ expression following primary in vitro activation and polarization of naive mouse CD4⁺ T cells under Th1- and Th2-promoting conditions. Naïve T cells (99% CD4⁺, 99% l-selectin⁺) were purified from spleens of BALB/c mice and activated under Th1- or Th2-promoting conditions (see Materials and Methods). Before activation and at 3, 5, 7, 9 and 12 days after activation, duplicate samples were stained with DATK32 antibody (anti-α₄/β₇) and analysed by flow cytometry. The mean percentage of α₄/β₇⁺ cells is shown at each time-point. Error bars represent standard deviations of duplicate samples. In all cases, ≤ 1% of cells were stained with an isotype control antibody.