Glutamine depletion potentiates leucocyte-dependent inflammatory events induced by carrageenan or Clostridium difficile toxin A in rats

Abstract

This research investigated the effect of glutamine (Gln) depletion on leucocyte-dependent inflammatory events. Rats were treated intraperitoneally, 16 hr prior to the peak of every parameter evaluated, with either 0.9% NaCl, methionine-sulphoximine (MSO, an inhibitor of endogenous Gln synthesis, 25 mg/kg) or with MSO + Gln (MSO as above plus Gln 3 g/kg in three doses). MSO-induced Gln depletion increased paw oedema induced both by carrageenan (Cg) and by Clostridium difficile toxin A (TxA) (66.2% and 45.5%, respectively; P < 0.05). In dextran-injected animals, oedema and myeloperoxidase (MPO) activity were not modified by Gln depletion. In Cg-treated paws, Gln depletion increased MPO activity by 44% (P < 0.05), interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) concentrations by 47% and 52%, respectively (P < 0.05), and immunostaining for TNF-alpha in paw tissue. In TxA-injected paws, Gln depletion increased MPO activity (46%; P < 0.05). Gln depletion increased Cg- and TxA-induced neutrophil migration to subcutaneous air pouches by 56% and 77% (P < 0.05), respectively, but did not affect migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP). Gln infusions reversed all the effects of MSO. Leucocyte counts did not differ between groups. Gln depletion potentiates acute inflammation, possibly by increasing neutrophil migration through resident cell activation and production of IL-1beta and TNF-alpha. Gln supplementation reverses these effects and may be useful during inflammatory catabolic stress.

Figure 1

Effect of methionine-sulphoximine (MSO) on plasma glutamine concentration. (a) MSO (25 mg/kg, i.p) was administered at 0 hr and blood was withdrawn for determination of plasma glutamine concentration by HPLC at 0, 8, 16 and 24 hr. (b) MSO (25 mg/kg, i.p) was administered at 0 hr to the MSO and MSO + Gln groups. Additionally, the MSO + Gln group received Gln (1 g/kg, i.p.) at 0, 6 and 12 hr. The saline group received only saline solution. Plasma Gln concentration was determined by HPLC 16 hr after the first injections. The dots and bars represent the mean ± SEM of plasma Gln concentrations, in mm, of five or six animals per treatment. *P < 0·05 versus 0 hr (a) and *P < 0·05 versus the saline group (b) using anova/Bonferroni.

Figure 2

Effect of methionine-sulphoximine (MSO) with or without glutamine (Gln) supplementation on paw oedema induced by carrageenan (Cg), Clostridium difficile toxin A (TxA) or dextran (Dx). MSO (25 mg/kg, i.p.) was administered at 0 hr to the MSO and MSO + Gln groups. Additionally, the MSO + Gln group received Gln (1 g/kg, i.p.) at 0, 6 and 12 hr. The saline and control groups received only saline solution i.p. Cg (100 μg) was injected at 12 hr (a), TxA (0·33 ng) at 7 hr (b) and Dx (100 μg) at 15·5 hr (c), to the hind paws of animals in the saline, MSO and MSO + Gln groups. The dots on the curve represent the mean ± SEM of the variation of paw volume. *P < 0·05 versus saline group using anova/Bonferroni.

Figure 3

Effect of methionine-sulphoximine (MSO) with or without glutamine (Gln) supplementation on myeloperoxidase (MPO) activity in paws injected with carrageenan (Cg) or Clostridium difficile toxin A (TxA). After termination of the Cg and TxA oedema experiments, samples of skin (a) and subcutaneous tissue (b) of the paws were harvested for determination of MPO activity. The bars represent the mean ± SEM of units of MPO per 5 mg of tissue. *P < 0·05 versus the saline group using anova/Bonferroni.

Figure 4

Effect of methionine–sulphoximine (MSO) with or without glutamine (Gln) supplementation on neutrophil migration to subcutaneous air pouches induced by carrageenan (Cg), Clostridium difficile toxin A (TxA) or FMLP. MSO (25 mg/kg, i.p.) was administered at 0 hr to the MSO and MSO + Gln groups. In addition, the MSO + Gln group received Gln (1 g/kg, i.p.) at 0, 6 and 12 hr. The saline and control groups received only saline solution i.p. Neutrophil migration was induced by injection of Cg (300 μg;a), TxA (0·1 ng;b) or fMLP (44 ng; c) at 12, 6 and 12 hr, respectively, to animals of the saline, MSO and MSO + Gln groups. The control group received only saline solution in the air pouch. At 16 hr, animals were killed and the leucocytes that had migrated to the air pouch were counted. The bars represent the mean ± SEM of the number of neutrophils that migrated in five or six animals per treatment. *P < 0·05 versus the saline group using anova/Bonferroni.

Figure 5

Effect of methionine-sulphoximine (MSO) with or without glutamine (Gln) supplementation on TNF-α detection by immunohistochemistry in paw tissue treated with carrageenan (Cg). MSO (25 mg/kg, i.p.) was administered at 0 hr to the MSO and MSO + Gln groups. Additionally, the MSO + Gln group received glutamine (Gln 1 g/kg, i.p.) at 0, 6 and 12 hr. The saline and control groups received only saline solution i.p. At 12 hr, Cg (100 μg, subplantar) was injected into animals in the saline (b), MSO (c) and MSO + Gln (d) groups and saline solution was injected into control animals (a). One hour later skin and subcutaneous tissue of the ventral region of the paws were harvested for TNF-α immunohistochemistry. In the MSO group (c), there are more cells labelled for TNF-α than in the saline group (b). Gln administration (d) caused the number of TNF-α labelled cells to decrease to levels compatible with those seen in the saline group (b).