Histamine-induced inhibition of leukotriene biosynthesis in human neutrophils: involvement of the H2 receptor and cAMP

Abstract

1. Histamine is generally regarded as a pro-inflammatory mediator in diseases such as allergy and asthma. A growing number of studies, however, suggest that this autacoid is also involved in the downregulation of human polymorphonuclear leukocyte (PMN) functions and inflammatory responses through activation of the Gs-coupled histamine H(2) receptor. 2. We report here that histamine inhibits thapsigargin- and ligand (PAF and fMLP)-induced leukotriene (LT) biosynthesis in human PMN in a dose-dependent manner. 3. The suppressive effect of histamine on LT biosynthesis was abrogated by the histamine H(2) receptor antagonists cimetidine, ranitidine, and tiotidine. In contrast, the histamine H(1), H(3), and H(4) receptor antagonists used in this study were ineffective in counteracting the inhibitory effect of histamine on the biosynthesis of LT in activated human PMN. 4. The inhibition of LT biosynthesis by histamine was characterized by decreased arachidonic acid release and 5-lipoxygenase translocation to the nuclear membrane. 5. Incubation of PMN with the cAMP-dependent protein kinase (PKA) inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide prevented the inhibitory effect of histamine on LT biosynthesis, suggesting an important role for PKA in this effect of histamine on LT biosynthesis in PMN. 6. These data provide the first evidences that, similarly to adenosine and prostaglandin E(2), histamine is a potent suppressor of LT biosynthesis, and support the concept that histamine may play a dual role in the regulation of inflammation.

Figure 1

Effect of histamine on LT biosynthesis in activated human PMN. Pre-warmed PMN suspensions (5 × 10⁶ cells ml⁻¹, 37°C) were stimulated with PAF, fMLP, or thapsigargin, as described in Methods. Incubations were terminated by adding 0.5 volume of a cold (4°C) stop solution containing 12.5 ng of both 19-OH-PGB₂ and PGB₂ as internal standards, and 5-LO products were analyzed as described in Methods. Histamine was added to the cell suspensions 5 min before the addition of the stimuli. The data shown are the mean (±s.e.m.) of three experiments, each performed in triplicates.

Figure 2

Effect of histamine receptor antagonists on the histamine-induced inhibition of LT biosynthesis. Pre-warmed PMN suspensions (5 × 10⁶ cells ml⁻¹, 37°C) were stimulated with 100 nM thapsigargin, as described in Methods. Incubations were terminated by adding 0.5 volume of a cold (4°C) stop solution containing 12.5 ng of both 19-OH-PGB₂ and PGB₂ as internal standards and analyzed for 5-LO products, as described in Methods. The histamine receptor antagonists and histamine (1 μM) were always added to the cell suspensions 10 and 5 min, respectively, before the addition of thapsigargin. Data shown are the mean (±s.e.m.) of at least three experiments, each performed in triplicates.

Figure 3

Effect of histamine on AA release in stimulated human PMN. (a) Pre-warmed PMN suspensions (10⁷ cells ml⁻¹; 37°C) were pre-incubated for 5 min in the presence of either 1 μM histamine or the H₂R agonist amthamine (or diluent), then stimulated with 100 nM thapsigargin for the indicated times. (b) Pre-warmed PMN suspensions (10⁷ cells ml⁻¹; 37°C) were pre-incubated for 15 min in the presence of 10 μM H-89 (or diluent), followed by a further 5 min pre-incubation with 1 mM histamine. PMN were then stimulated with 300 nM PAF (or diluent) for 2 min. All incubations (a, b) were stopped by adding 1 volume of an ice-cold (4°C) stop solution containing 12.5 ng of both 19-OH-PGB₂ and PGB₂, and 20 ng ²H₈-AA. AA was extracted and purified from the denatured incubation media by RP-HPLC using an on-line extraction procedure, and analyzed by LC-MS, as described in Methods. The data shown (a, b) are the mean (±s.e.m.) of duplicate incubations from single experiments representative of three.

Figure 4

Effect of CGS-21680 and histamine on cPLA₂ and 5-LO translocation. Pre-warmed GMCSF/TNF-α/cytochalasin B-treated PMN suspensions (10⁷ cells ml⁻¹, 37°C) were incubated with 10 μM CGS-21680 or 1 mM histamine, and then stimulated with fMLP, as described in Methods. Incubations were stopped by the addition of 1 volume of cold (4°C) incubation buffer, and the cell suspensions were immediately centrifuged (4°C). Fractionation of the cell pellets was performed as described in Methods, and membrane fractions were analyzed by SDS–PAGE and immunoblotted with the cPLA₂ and the 5-LO antibodies. Data shown are from one experiment representative of two.

Figure 5

Effect of histamine and EGTA on Ca²⁺ mobilization in human PMN. Fura-2-loaded PMN (10⁷ cells ml⁻¹) were pre-incubated at 37°C for 15 min, and then stimulated with 100 nM PAF (a, b). The PKA inhibitor H-89 (10 μM) and histamine (10 μM) were added 10 and 5 min, respectively, before PAF stimulation and histamine. Additions of PAF were performed 10 s after the beginning of data acquisition. Fluorescence was measured at excitation and emission wavelengths of 340 and 510 nm, respectively, as described in Methods. In experiments where chelation of extracellular Ca²⁺ was performed (b), EGTA (2 mM) was added simultaneously with PAF. Data shown are from single experiments representative of at least three.

Figure 6

(a) Effect of EGTA on agonist-induced LT biosynthesis in human PMN. Pre-warmed PMN suspensions (5 × 10⁶ cells ml⁻¹, 37°C) were stimulated in the presence or absence of 2 mM EGTA, as described in Methods. (b) Inhibitory effect of histamine on LT biosynthesis in the presence of EGTA. Pre-warmed PMN suspensions (5 × 10⁶ cells ml⁻¹, 37°C) were stimulated as described in Methods, in the presence or absence of 2 mM EGTA. Histamine (1 mM) was added to the cell suspensions 5 min before the addition of PAF. All incubations were terminated by adding 0.5 volume of a cold (4°C) stop solution containing 12.5 ng of both 19-OH-PGB₂ and PGB₂ as internal standards and 5-LO products were analyzed, as described in Methods. In incubations where chelation of extracellular Ca²⁺ was performed, EGTA was added simultaneously with either PAF, fMLP, or thapsigargin. Data represent the mean (±s.e.m.) of triplicate incubations from a single experiment representative of three.