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. 2009 Aug;157(7):1232-40.
doi: 10.1111/j.1476-5381.2009.00195.x. Epub 2009 Jun 22.

Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kappaB signal transduction pathways

Pilar Requena  1 , Abdelali Daddaoua, Emilia Guadix, Antonio Zarzuelo, María Dolores Suárez, Fermín Sánchez de Medina, Olga Martínez-Augustin
Affiliations

Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-kappaB signal transduction pathways

Pilar Requena et al. Br J Pharmacol. 2009 Aug.

Abstract

Background and purpose: Bovine glycomacropeptide (BGMP) is a natural milk peptide that is produced naturally in the gastrointestinal tract during digestion. Glycomacropepide has intestinal anti-inflammatory activity, but the mechanism of action is unknown. Here we have characterized the effects of BGMP on monocytes.

Experimental approach: We have used human THP-1 cells as an in vitro monocyte model. The effect of BGMP on the secretion of tumour necrosis factor (TNF), interleukin (IL)-1beta and IL-8 was assessed, as well as the involvement of the NF-kappaB and MAP kinase signalling pathways. The stimulatory effect of BGMP was also tested in human peripheral blood monocytes.

Key results: BGMP up-regulated the secretion of TNF, IL-1beta and IL-8 in a concentration-dependent fashion. The biological activity was exerted by the intact peptide, because cytokine secretion was not affected by protease inhibitors. The secretion of IL-8 and specially TNF and IL-1beta was blocked by PD98059, SP600125, SB203580 and Bay11-7082, suggesting the involvement of the MAP kinases p38, c-Jun N-terminal kinase and ERK and particularly the NF-kappaB pathway, although IL-8 secretion was independent of p38. BGMP was shown to elicit the phosphorylation of IkappaB-alpha and the nuclear translocation of the NF-kappaB subunits p50 and p65. The effect of BGMP on cytokine secretion was validated in human primary blood monocytes.

Conclusions and implications: BGMP stimulates human monocytes, operating via MAP kinase and NF-kappaB pathways. BGMP may exert an indirect intestinal anti-inflammatory effect by potentiating host defences against invading microorganisms.

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Figures

Figure 2
Figure 2
Effect of bovine serum albumin (BSA) and bovine glycomacropeptide (BGMP) on the production of tumour necrosis factor (TNF), interleukin (IL)-1β and IL-8 by THP-1 cells. After a 24 h incubation with either peptide (1 mg·mL−1) the secretion of cytokines was measured in foetal bovine serum (FBS)-containing or FBS-free culture medium by ELISA. Results are expressed as mean ± SEM of three different experiments (n= 3 in each experiment). *P < 0.05 versus control (C).
Figure 1
Figure 1
Concentration–response curves for the production of tumour necrosis factor (TNF), interleukin (IL)-1β and IL-8 by THP-1 cells in the presence of bovine glycomacropeptide (BGMP). After a 24 h incubation in the presence of the products, secretion of cytokines was measured in foetal bovine serum-containing culture medium by ELISA. Results are expressed as mean ± SEM of three different experiments (n= 3 in each experiment). *P < 0.05 versus control.
Figure 3
Figure 3
Effect of protease inhibitors on the production of tumour necrosis factor (TNF), interleukin (IL)-1β and IL-8 by THP-1 cells stimulated with bovine glycomacropeptide (BGMP). After 1 h of incubation with the serine protease inhibitor Pefabloc® (PEF, 0.1 mmol·L−1) or a protease inhibitor cocktail (PI, 1:200 v·v−1) the cells were exposed to BGMP (1 mg·mL−1). After a 24 h incubation, the secretion of cytokines was measured in the culture medium by ELISA. Results are expressed as mean ± SEM of three different experiments (n= 3 in each experiment). Means marked with the same letter were not different from each other, but were different from means marked with a different letter; P < 0.05. C, control group.
Figure 4
Figure 4
Effect of MAP kinase and NF-κB inhibitors on tumour necrosis factor (TNF), interleukin (IL)-1β and IL-8 secretion by THP-1 cells stimulated with bovine glycomacropeptide (BGMP). Cytokine concentrations were determined by ELISA in the supernatants of cells pre-incubated for 1 h with the signal transduction inhibitors [MAP kinase inhibitors PD98059 (ERK1/2 inhibitor), SB203580 (ERK1/2 inhibitor), SP600125 (JNK inhibitor) or the NF-κB inhibitor Bay 11-7082] followed by a 24 h incubation with BGMP. Data are mean ± SEM from at least three independent experiments. Means marked with the same letter were not different from each other, but were different from means marked with a different letter; P < 0.05. C, control group.
Figure 5
Figure 5
Effect of bovine glycomacropeptide (BGMP) on the phosphorylation of IκB-α (A) and on the translocation of p50 and p65 to the nucleus of THP-1 cells (B). (A) Cells were cultured with BGMP for different periods of time and Western blots were carried out with cell extracts. α-Actin was used as a loading control. (B) Cells were cultured for different periods with BGMP or BGMP plus the NF-κB inhibitor Bay 11-7082 (BAY). Western blot experiments were carried out with nuclear extracts.
Figure 6
Figure 6
Concentration–response curves for the production of tumour necrosis factor (TNF), interleukin (IL)-1β and IL-8 by primary human monocytes in the presence of BGMP. After a 24 h incubation in the presence of the products, secretion of cytokines was measured in the culture medium by ELISA. Logistic sigmoidal curves could be fitted in the cases of TNF and IL-8 (EC50 0.92 ± 0.09 mg·mL−1 and 0.72 ± 0.40 µg·mL−1 respectively). Results are expressed as mean ± SEM of two different experiments (n= 3 in each experiment). *P < 0.05 versus control.
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