Tumour necrosis factor-alpha and leukotriene B(4) mediate the neutrophil migration in immune inflammation

Abstract

1. We investigated the mediators responsible for neutrophil migration induced by ovalbumin (OVA) in immunized mice and the mechanisms involved in their release. 2. OVA administration promoted dose- and time-dependent neutrophil migration in immunized, but not in non-immunized mice, which was mediated by leukotriene B(4) (LTB(4)) and tumour necrosis factor (TNF)alpha, since it was inhibited by LTB(4) synthesis inhibitor (MK 886) or by LTB(4) receptor antagonist (CP 105,696), by dexamethasone and by antiserum to TNFalpha (82, 85, 63 and 87%, respectively). Confirming TNFalpha involvement, OVA challenge in immunized p55 TNF receptor deficient mice (p55(-/-)) did not promote neutrophil migration (control: 2.90 +/- 0.68; p55(-/-): 0.92+/-0.23 x 10(6) neutrophils cavity(-1)). 3. OVA-stimulated peritoneal cells from immunized mice released a neutrophil chemotactic factor which mimicked, in naive mice, neutrophil migration induced by OVA. 4. Supernatant chemotactic activity is due to TNFalpha and LTB(4), since its release was inhibited by MK 886 (93%) and dexamethasone (90%), and significant amounts of these mediators were detected. 5. TNFalpha and LTB(4) released by OVA challenge seem to act through a sequential mechanism, since MK 886 inhibited (88%) neutrophil migration induced by TNFalpha. Moreover, peritoneal cells stimulated with TNFalpha released LTB(4). 6. CD(4)(+) T cells are responsible for TNFalpha release, because the depletion of this subset prevented the release of TNFalpha (control: 400 +/- 25; immunized: 670 +/- 40; CD(4)(+) depleted: 435 +/- 18 pg ml(-1)). 7. In conclusion, neutrophil migration induced by OVA depends on TNFalpha released by CD(4)(+) cells, which acts through an LTB(4)-dependent mechanism.

Figure 1

Dose-dependence and time-course of neutrophil migration induced by OVA. (A) OVA was injected at the indicated doses into the peritoneal cavity of non-immunized (c; control) or immunized animals and neutrophil migration was determined 4 h later. Neutrophil migration was also evaluated when 10 μg of KLH was injected into immunized mice. (B) Time-course of the neutrophil migration induced by OVA (10 μg cavity⁻¹) in control or in immunized mice. Data are mean±s.e.mean. * P<0.05 compared to the respective controls (ANOVA followed by Bonferroni t-test). Results are representative of two separate experiments with six mice per group.

Figure 2

Effect of anti-inflammatory drugs on OVA-induced neutrophil migration. Immunized animals were treated with PBS (s.c.; 30 min before), indomethacin (indo; 5 mg kg⁻¹; s.c.; 30 min before), L-N^G-nitroarginine (nitro; 50 mg kg⁻¹; s.c.; 30 min before), meclizine (mec; 20 mg kg⁻¹; s.c; 30 min before), BN 50730 (BN; 10 mg kg⁻¹; s.c.; 30 min before), MK 886 (MK; 1 mg kg⁻¹; orally; 1 h before), CP 105,696 (CP; 3 mg kg⁻¹; s.c.; 30 min before) or dexamethasone (dexa; 1 mg kg⁻¹; s.c.; 1 h before) and then challenged with OVA (10 μg cavity⁻¹). The first bar represents the neutrophil migration induced by PBS injected i.p. (C). Neutrophil migration was evaluated 4 h after OVA challenge. The values are mean±s.e.mean. *P<0.05 compared to PBS i.p. group, and #P<0.05 compared to PBS treated OVA-injected group (ANOVA followed by Bonferroni t-test) treatment. Results are representative of three separate experiments with five mice per group.

Figure 3

Inhibition of neutrophil migration by anti-TNFα serum treatment and in p55^−/− mice. (A) BALB/c immunized mice were injected with control serum (α-C) or with anti-TNFα serum (35 μl cavity⁻¹) 15 min before the challenge with OVA (10 μg cavity⁻¹). Control (c) mice were injected with OVA and immunized mice were injected with OVA or vehicle (0.5 ml cavity⁻¹), and the neutrophil migration evaluated 4 h later. (B) Control and immunized C57BL/6 (black bars) or p55^−/− (white bars) were challenged with OVA (10 μg cavity⁻¹), and 4 h later the neutrophil migration was estimated. Data are mean±s.e.mean. *P<0.05 vs non-immunized control, and #P<0.05 compared to α-C and immunized C57BL/6 (ANOVA followed by Bonferroni t-test). Results are representative of two separate experiments with five mice per group.

Figure 4

Effect of MK 886 on neutrophil migration induced by TNFα, LTB₄ or fMLP. Neutrophil migration was induced by TNFα (40 ng cavity⁻¹), LTB₄ (25 ng cavity⁻¹) or fMLP (11 μg cavity⁻¹) in mice pre-treated with PBS (striped bars) or MK 886 (1 mg kg⁻¹; black bar). Control mice received only PBS i.p. (white bar). Neutrophil migration was evaluated 4 h after the stimuli or PBS injection. Data are mean±s.e.mean. *P<0.05 compared to PBS group, and #P<0.05 compared to TNFα group (ANOVA followed by Bonferroni t-test). Results are representative of two separate experiments with five mice per group.

Figure 5

Release of a neutrophil chemotactic factor by OVA-stimulated peritoneal cells. (A) Neutrophil migration was induced in naive animals by the i.p. administration of 1 ml of peritoneal cell supernatant obtained from control (c) or immunized mice, which were stimulated with OVA or KLH at the indicated concentrations for 1 h, washed and cultured for 6 h. (B) Neutrophil migration induced by OVA (10 μg ml⁻¹)-stimulated peritoneal cell supernatant obtained from control (c) or immunized mice, incubated for the times indicated (h). Neutrophil migration was quantified 4 h after the supernatant injections and the values are expressed as mean±s.e.mean. *P<0.05 compared to respective control groups (ANOVA followed by Bonferroni t-test). Results are representative of two separate experiments with five mice per group.

Figure 6

Effect of anti-inflammatory drugs on the release of the neutrophil chemotactic factor. Peritoneal cells were stimulated with OVA (10 μg ml⁻¹) for 1 h, washed and incubated for a further 6 h. The cells were cultured in the absence (PBS) or in the presence of indomethacin (indo; 10 μM), L-N^G-nitroarginine (nitro; 100 μM), meclizine (mec; 100 μM), BN 50730 (BN; 100 μM), MK 886 (MK; 1 μM) or dexamethasone (dexa; 10 μM). The supernatants were injected into the peritoneal cavities (1 ml) of naive mice and neutrophil migration was quantified 4 h later. Data are presented as mean±s.e.mean. *P<0.05 compared to the PBS treatment (ANOVA followed by Bonferroni t-test). Results are representative of two separate experiments with five mice per group.

Figure 7

Effect of lymphocyte depletion on the production of neutrophil chemoattractant activity. (A) Neutrophil migration was induced in naive mice by the i.p. administration of 1 ml of supernatant of peritoneal cells obtained from control (c) or immunized mice depleted of T or B cells. (B) Neutrophil migration was induced in naive mice by the i.p. administration of 1 ml of peritoneal cell supernatant obtained from control (c) or immunized mice depleted of CD₄⁺ and CD₈⁺ T subsets. CD₄⁺ T cell depleted peritoneal cell suspension was incubated for 2 h with OVA, washed and then reconstituted with CD₄⁺ T cells (CD₄⁺ R) obtained from immunized mice (striped bar). Depletion was performed as described in Methods. Data are mean±s.e.mean. *P<0.05 compared to control group and #P<0.05 compared to non-depleted group (ANOVA followed by Bonferroni t-test). Results are representative of two separate experiments with five mice per group.

Figure 8

Representative flow cytometry histograms for CD₄ expression on peritoneal cell suspension before and after immuno-magnetic depletion. Peritoneal cells were analysed for cell-surface expression of CD₄ using L3T4 FITC-conjugated monoclonal antibody. (A) Peritoneal cell suspension incubated with isotype matched antibody (control). Peritoneal cell suspension (B) or depleted CD₄⁺ peritoneal cell suspension (C) were incubated with FITC-conjugated L3T4. M1 indicates the region of CD4-expressing cells. Histograms shown are from one of three separate experiments and are expressed in log scale (analysis of 10,000 cells).