Allergenic sensitization prevents upregulation of haemopoiesis by cyclo-oxygenase inhibitors in mice

Abstract

1. We evaluated whether immunization affects bone-marrow responses to indomethacin, because allergenic sensitization and challenge upregulate responses to haemopoietic cytokines (including IL-5-driven eosinopoiesis) in murine bone-marrow, while indomethacin upregulates haemopoiesis and protects bone-marrow from radiation damage. 2. Progenitor (semi-solid) and/or precursor (liquid) cultures were established from bone-marrow of: (a) normal mice; (b) ovalbumin-sensitized mice, with or without intranasal challenge. Cultures were established with GM-CSF (2 ng ml(-1)) or IL-5 (1 ng ml(-1)), respectively, alone or associated with indomethacin (10(-7) - 10(-11) M) or aspirin (10(-7) - 10(-8) M). Total myeloid colony numbers and numbers of eosinophil-peroxidase-positive cells were determined at day 7. 3. In naïve BALB/c mice, indomethacin (10(-7) - 10(-9) M) increased GM-CSF-stimulated myeloid colony formation (P=0.003 and P=0.009, respectively). In contrast, it had no effect on bone-marrow of ovalbumin-sensitized and challenged mice. Indomethacin (10(-7) - 10(-9) M) also increased eosinophil precursor responses to IL-5 in bone-marrow of naïve (P<0.001 and P=0.002 respectively), but not sensitized-challenged mice. Aspirin (10(-7) M) had similar effects, equally abolished by sensitization. Enhancement of haemopoiesis by indomethacin required adherent cells from naïve bone-marrow. Nonadherent cells responded to IL-5 but not to indomethacin. Indomethacin was effective on bone-marrow from sham-sensitized, ovalbumin-challenged, but not from sensitized, saline-challenged mice. Plasma transfer from immune mice abolished eosinophil precursor responses to indomethacin in bone-marrow of naïve recipients. This was not prevented by previous removal of antibody from immune plasma. 4. COX inhibitors enhance haemopoiesis in naïve but not allergic mice. Responsiveness to indomethacin can be abolished either by active sensitization or by immune plasma transfer. Specific antibody is not involved.

Figure 1

Effect of indomethacin on GM-CSF-induced colony formation. The data are means±s.e.mean of the number of myeloid colonies formed by bone-marrow from naïve (A) and from sensitized-challenged (B) BALB/c mice, in the presence of GM-CSF (2 ng ml⁻¹), alone, or in association with indomethacin, at the indicated concentrations. Data are derived from 3 – 9 experiments. *Indicates significant differences relative to the GM-CSF controls (P=0.003 and P=0.009, respectively to 10⁻⁷ M and 10⁻⁹ M indomethacin).

Figure 2

Effect of indomethacin on IL-5-driven eosinophil differentiation. The data are means±s.e.mean of the per cent of EPO+cells present at day 7 in liquid cultures with bone-marrow cells from naïve (A) and from sensitized-challenged (B) BALB/c mice, in the presence of IL-5 (1 ng ml⁻¹), alone, or in association with indomethacin, at the indicated concentrations. Data are derived from 3 – 22 experiments. *Indicates significant differences relative to the IL-5 controls (P<0.001 for both concentrations). **Indicates significant difference relative to the IL-5 control of naïve mice (A) (P=0.016). In B, no significant differences were observed between indomethacin-treated and control cultures.

Figure 3

Effect of adherent cell depletion on bone-marrow response to indomethacin. Data are means±s.e.mean of the per cent of EPO+ cells present at day 7 in liquid cultures with bone-marrow cells from naïve BALB/c mice, in the presence of IL-5 (1 ng.ml⁻¹) alone, or in association with indomethacin at 10⁻⁷ – 10⁻¹¹ M (A) or at 10⁻⁷ M (B). In A, total (unseparated) or non-adherent bone-marrow cells were cultured. In B, cultures were established with a mixture of nonadherent cells (NAdh C) and adherent cells (Adh C) purified from naïve (N) or immune (I) donors. Data for A are derived from 3 – 8 experiments, and for B from four experiments. *Indicates significant differences to the IL-5 control (P<0.038).

Figure 4

Effect of indomethacin on IL-5-driven eosinophil differentiation. The data are means±s.e.mean of the per cent EPO+ cells present at day 7 in liquid cultures with bone-marrow cells from sensitized/sham-challenged (A) and from sham-sensitized/challenged (B) BALB/c mice, in the presence of IL-5 (1 ng ml⁻¹), alone, or in association with indomethacin, at the indicated concentrations. Data are derived from five experiments. *Indicates significant differences relative to the IL-5 controls (P<0.001 for both concentrations of indomethacin).

Figure 5

Depletion of antibody from immune plasma. (A) Data are OD₄₀₅ readings from the indicated dilutions of immune plasma, naïve plasma and Protein A-Sepharose-absorbed immune plasma, assayed in ELISA for ovalbumin-specific IgG antibodies, as described in Methods. (B), immuno-blot and (C), Ponceau Red-stained blot of ‘P' plasma from ovalbumin-sensitized and challenged mice; ‘A' absorbed immune plasma; ‘E' eluate from the last absorption cycle; shown as controls, ‘G' goat antimouse IgG (H+L) polyclonal antibody and ‘I' monoclonal mouse IgG.

Figure 6

Effect of plasma transfer on bone-marrow response to indomethacin. The data are means±s.e.mean of the per cent of EPO+cells present at day 7 in liquid cultures with total bone-marrow cells from naïve BALB/c mice, in the presence of IL-5 (1 ng ml⁻¹) alone, or in association with indomethacin (10⁻⁷ M). Mice were injected with plasma from naïve (N) or immune (I) donors. Plasma was absorbed as described in Methods to eliminate antibodies (+) or left untreated (−). Data are derived from 3 – 8 experiments. *Indicates significant differences to the IL-5 control (P=0.005).