Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response

Abstract

The local production of proinflammatory cytokines mediates the host response to inflammation, infection, and injury, whereas an overexpression of these mediators can injure or kill the host. Recently, we identified a class of multivalent guanylhydrazone compounds that are effective inhibitors of proinflammatory cytokine synthesis in monocytes/macrophages. The structure of one such cationic molecule suggested a molecular mimicry with spermine, a ubiquitous endogenous biogenic amine that increases significantly at sites of inflammation and infection. Here, we addressed the hypothesis that spermine might counterregulate the innate immune response by downregulating the synthesis of potentially injurious cytokines. When spermine was added to cultures of human peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), it effectively inhibited the synthesis of the proinflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, MIP-1alpha, and MIP-1beta. The inhibition of cytokine synthesis was specific and reversible, with significant inhibition of TNF synthesis occurring even when spermine was added after LPS. The mechanism of spermine-mediated cytokine suppression was posttranscriptional and independent of polyamine oxidase activity. Local administration of spermine in vivo protected mice against the development of acute footpad inflammation induced by carrageenan. These results identify a distinct molecular counterregulatory role for spermine in downregulating the monocyte proinflammatory cytokine response.

Figure 1

Spermine inhibits TNF synthesis from LPS-stimulated human PBMCs. Human PBMCs were exposed to the concentrations of spermine as indicated for 1 h, then stimulated by addition of LPS (100 ng/ml) and IFN-γ. TNF released into the conditioned supernatants collected after 4 h was measured by ELISA and results expressed as percentage of control (no spermine). Data shown are mean ± SEM of three independent experiments. (Control TNF = 7416 ± 763 pg/ml.)

Figure 2

Spermine suppression of TNF and MIP-1α is posttranscriptional in LPS-stimulated human PBMCs. Northern blots for TNF and MIP-1α in human PBMCs performed 4 h after stimulation by LPS (100 ng/ml). Where indicated, spermine (35 μM) was added 1 h before LPS. Results are representative of two separate experiments using 6 μg of total RNA applied per lane.

Figure 3

Spermine inhibition of TNF and MIP-1α protein in cell lysates and supernatants.Human PBMCs were exposed to spermine for 1 h, stimulated with LPS, and after 4 h of stimulation lysed as described in Materials and Methods. The levels of TNF and MIP-1α in the supernatants (a and c) and corresponding whole-cell lysates (b and d) are shown. Data are mean ± SEM.

Figure 3

Spermine inhibition of TNF and MIP-1α protein in cell lysates and supernatants.Human PBMCs were exposed to spermine for 1 h, stimulated with LPS, and after 4 h of stimulation lysed as described in Materials and Methods. The levels of TNF and MIP-1α in the supernatants (a and c) and corresponding whole-cell lysates (b and d) are shown. Data are mean ± SEM.

Figure 3

Spermine inhibition of TNF and MIP-1α protein in cell lysates and supernatants.Human PBMCs were exposed to spermine for 1 h, stimulated with LPS, and after 4 h of stimulation lysed as described in Materials and Methods. The levels of TNF and MIP-1α in the supernatants (a and c) and corresponding whole-cell lysates (b and d) are shown. Data are mean ± SEM.

Figure 3

Spermine inhibition of TNF and MIP-1α protein in cell lysates and supernatants.Human PBMCs were exposed to spermine for 1 h, stimulated with LPS, and after 4 h of stimulation lysed as described in Materials and Methods. The levels of TNF and MIP-1α in the supernatants (a and c) and corresponding whole-cell lysates (b and d) are shown. Data are mean ± SEM.

Figure 4

Spermine inhibition of MIP-1β, IL-1β, and IL-6, but not TGF-β from human PBMCs. Human PBMCs were pretreated with spermine for 60 min, then stimulated with LPS (100 ng/ml). 4 h after LPS treatment, supernatants were assayed by ELISA for: a MIP-1β; b IL-1β; c IL-6; and d TGF-β. Data are mean ± SEM of three experiments. Control cytokine levels: MIP-1β, 19 ng/ml; IL-1β, 32 pg/ml; IL-6, 6,000 pg/ml; and TGF-β, 680 ± 133 pg/ml.

Figure 4

Spermine inhibition of MIP-1β, IL-1β, and IL-6, but not TGF-β from human PBMCs. Human PBMCs were pretreated with spermine for 60 min, then stimulated with LPS (100 ng/ml). 4 h after LPS treatment, supernatants were assayed by ELISA for: a MIP-1β; b IL-1β; c IL-6; and d TGF-β. Data are mean ± SEM of three experiments. Control cytokine levels: MIP-1β, 19 ng/ml; IL-1β, 32 pg/ml; IL-6, 6,000 pg/ml; and TGF-β, 680 ± 133 pg/ml.

Figure 4

Spermine inhibition of MIP-1β, IL-1β, and IL-6, but not TGF-β from human PBMCs. Human PBMCs were pretreated with spermine for 60 min, then stimulated with LPS (100 ng/ml). 4 h after LPS treatment, supernatants were assayed by ELISA for: a MIP-1β; b IL-1β; c IL-6; and d TGF-β. Data are mean ± SEM of three experiments. Control cytokine levels: MIP-1β, 19 ng/ml; IL-1β, 32 pg/ml; IL-6, 6,000 pg/ml; and TGF-β, 680 ± 133 pg/ml.

Figure 4

Spermine inhibition of MIP-1β, IL-1β, and IL-6, but not TGF-β from human PBMCs. Human PBMCs were pretreated with spermine for 60 min, then stimulated with LPS (100 ng/ml). 4 h after LPS treatment, supernatants were assayed by ELISA for: a MIP-1β; b IL-1β; c IL-6; and d TGF-β. Data are mean ± SEM of three experiments. Control cytokine levels: MIP-1β, 19 ng/ml; IL-1β, 32 pg/ml; IL-6, 6,000 pg/ml; and TGF-β, 680 ± 133 pg/ml.

Figure 5

Spermine suppression of TNF synthesis in serum-free media and in the presence of polyamine oxidase inhibition. (a) PBMCs were cultured in serum-free media (OPTI-MEMI), stimulated with LPS for 4 h, then TNF levels in the media measured by ELISA as described. (b) Human PBMCs were cultured in RPMI-1640 containing FBS (10%) with the addition of aminoguanidine (0, 1, or 2 mM) as shown. 4 h after LPS treatment, conditioned supernatants were assayed for TNF by ELISA as described above.The data shown are from a representative experiment (repeated twice with similar results). Each point represents mean ± SEM from six replicates.

Figure 5

Spermine suppression of TNF synthesis in serum-free media and in the presence of polyamine oxidase inhibition. (a) PBMCs were cultured in serum-free media (OPTI-MEMI), stimulated with LPS for 4 h, then TNF levels in the media measured by ELISA as described. (b) Human PBMCs were cultured in RPMI-1640 containing FBS (10%) with the addition of aminoguanidine (0, 1, or 2 mM) as shown. 4 h after LPS treatment, conditioned supernatants were assayed for TNF by ELISA as described above.The data shown are from a representative experiment (repeated twice with similar results). Each point represents mean ± SEM from six replicates.

Figure 6

Spermine suppresses carrageenan-induced inflammation in mice. Carrageenan (0.2%) was injected into the left rear footpad of C3H/ HeN mice with the concentration of spermine indicated in 50 μl; ; vehicle (1× PBS) was injected into the right hindpaw. 28 h later, the footpad thickness was measured with a caliper, and the difference between right and left hindpaw determined. Data are mean ± SEM (mm) from three experiments, with n = 5 animals in each experimental group.