Haptoglobin directly affects T cells and suppresses T helper cell type 2 cytokine release

Abstract

T helper cell type 1 (Th1) and type 2 (Th2) immune responses are characterized by a different pattern of cytokine expression following T-cell activation. Alterations of the ratio of Th1 to Th2 cells are important determinants of susceptibility to viral and parasitic infections, allergies, anti-tumour responses, and autoimmunity. In this work we bring new evidence for an effect of haptoglobin (Hp), a positive acute-phase protein, on T-lymphocyte functions. We show that Hp specifically interacts with both resting and activated CD4+ and CD8+ T cells. This specific binding results in a strong suppression of induced T-cell proliferation. In addition, Hp exhibits a strong in vitro inhibitory effect on Th2 cytokine release, while the production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) is only slightly inhibited at high Hp doses. As a result, the presence of Hp promotes Th1 activation over Th2 activation in vivo as evidenced in Hp-deficient mice. Anti-CD3 monoclonal antibody injection indeed resulted in predominant IL-4 production in Hp-/- mice, in contrast to predominant IFN-gamma production in Hp+/+ mice. We conclude that Hp plays a modulating role on the Th1/Th2 balance by promoting a dominant Th1 cellular response. This points to a role of acute-phase proteins in balancing immune responses.

Figure 1

Hp inhibits T-cell proliferation. (a) Dose-dependent inhibitory effects of Hp on PHA-induced T-cell proliferation: 0·2 × 10⁶ PBMC/well were stimulated with PHA (5 μg/ml) in the presence of increasing Hp concentrations. T-cell proliferation was measured by [³H]thymidine incorporation assay at day 3. Results are shown as the mean ± SEM from three donors. (b) The inhibitory effect of Hp is compared to that of human serum albumin (HSA). One representative experiment is shown. (c) Hp1-1 and Hp2-1 similarly suppress T-cell proliferation following different stimulatory triggers; 0·5 × 10⁶ PBMC/ml were stimulated with either 5 μg PHA or 1 ng PMA/1 μg ionomycin for 3 days, or alternatively with 0·36 μg tetanus toxoid for 6 days, in the absence or presence of 500 μg of either Hp1-1 or Hp2-1. T-cell proliferation was evaluated by [³H]thymidine incorporation assay. Results represent the mean ± SEM in three independent experiments. (d) The effect of 500 μg/ml of Hp was evaluated on PHA-stimulated PBMC, T + NK, and T-cell cultures isolated from the same donors. The inhibition of T-cell proliferation was evaluated after a 72-h incubation and expressed as percentage of control cultures where no Hp was added. Results represent the mean ± SEM from three experiments. P > 0·05 for all comparisons between cultures.

Figure 2

Hp binds to resting and activated CD4⁺ and CD8⁺ T lymphocytes. CD4⁺ and CD8⁺ T cells (0·5 × 10⁶) were incubated overnight in the absence or presence of PMA + ionomycin (1 ng/ml + 1 μg/ml, respectively). After washing with PBS, cells were incubated with human serum albumin 0·1%/PBS for 15 min and washed with PBS again. Hp–FITC was added and cells were incubated for 30 min at 4°, washed and fixed with paraformaldehyde 1% in PBS. (a) Effect of increasing doses of Hp–FITC. Hp–FITC binding was assessed by flow cytometry and results were expressed as either percentage positive cells (upper panels) or mean fluorescence intensity (MFI) (lower panels) of 10⁴ of resting (○) or activated (•) T cells. (b) Representative histograms of binding of Hp–FITC to CD4 or CD8 T cells, either resting or activated. The figure shows autofluorescence, binding of Hp–FITC (dotted line), and inhibition of Hp–FITC binding by a 25-fold excess of unlabelled Hp (solid line).

Figure 3

Differential effects of Hp on T helper cytokine release; 0·5 × 10⁶ T cells in a final volume of 1 ml were cultured for 72 hr in the presence of hCD80-P815 cells (1 : 1 ratio) and 1 μg of anti-CD3 mAb. Hp was added at increasing doses (50, 250, 500 and 1000 μg). IL-2, IFN-γ, IL-4, IL-5, IL-10 and IL-13 release was measured using ELISA assays. To prevent IL-2 and IL-4 consumption, IL-2 and IL-4 receptors were blocked with mAbs (see the Materials and methods section). Results are shown as mean ± SEM from n = 4–6 experiments. *P < 0·05; **P < 0·01 versus control.

Figure 4

Hp affects the Th1/Th2 balance in an animal model of cytokine release syndrome; 10 μg of the 145-2C11 hamster anti-mouse CD3 mAb was administered intraperitoneally to 7–8-week-old Hp^+/+ and Hp^−/− male C57BL/6J mice. Two other groups of mice received 10 μg of LPS 36 hr prior to injection of the antibody. Blood was collected after 90 min. Serum IL-4 and IFN-γ levels were determined (a) using the ELISA technique and the ratio IFN-γ/IL-4 was calculated (b). Results are reported as the mean serum cytokine level of six mice per group from one representative out of three performed experiments with similar results. ***P < 0·001 vs. Hp^−/− versus Hp^+/+.