Elsevier

Biochemical Pharmacology

Volume 62, Issue 1, 1 July 2001, Pages 141-147
Biochemical Pharmacology

Involvement of nuclear factor-kappa B (NF-κB) activation in mitogen-induced lymphocyte proliferation: inhibitory effects of lymphoproliferation by salicylates acting as NF-κB inhibitors2

https://doi.org/10.1016/S0006-2952(01)00640-2Get rights and content

Abstract

The transcription factor nuclear factor-kappa B (NF-κB) is involved in the production of inflammatory cytokines and in the control of the inflammatory response. Some nonsteroidal anti-inflammatory drugs such as acetylsalicylic acid (ASA) or salicylate are known to exert some of their anti-inflammatory pharmacological properties independently of cyclooxygenase inhibition. For ASA and salicylate, an NF-κB inhibitory effect at mM concentrations (pharmacological plasma concentrations reached in vivo) has been shown. We studied the action of ASA, salicylate, and several NF-κB inhibitors on the mitogen-induced activation of peripheral blood lymphocytes (PBL) and purified T cells. We showed that ASA and salicylate (1–3 mM) (but not indomethacin, a specific cyclooxygenase inhibitor) as well as a group of chemically unrelated inhibitors of NF-κB (including the sesquiterpene lactone parthenolide, Bay 11-7082, sulfasalazine, the proteasome inhibitor MG-132 and the peptide SN-50, an inhibitor of the nuclear transfer of the p50 subunit of NF-κB), were potent inhibitors of phytohemoagglutinin-activated PBL and T cell proliferation. At the same concentrations, they inhibited NF-κB binding to DNA in nuclear extracts. The inhibition of proliferation was not relieved by exogenous interleukin (IL)-2. We concluded that NF-κB activation has a fundamental role in T cell proliferation independently of IL-2 production. Some pharmacological actions of ASA may be ascribed to the inhibition of immune cell proliferation via the inhibition of the transcription factor NF-κB.

Introduction

Many of ASA’s therapeutic effects are clearly due to the inhibition of prostaglandin synthesis [1]. However, not all of ASA’s anti-inflammatory actions can be accounted for by simple inhibition of COX 1 and 2. For example, therapeutic serum concentrations of salicylate, the major metabolite of ASA and a poor inhibitor of COX 1 and COX 2, correlate better with clinical anti-inflammation than serum concentrations of ASA [2], [3]. It was recently found that ASA and salicylate disrupt signal transduction by inhibiting the translocation to the nucleus of the transcriptional regulator NF-κB [4]. NF-κB is a dimeric protein complex composed of members of the Rel family of transcription factors and is necessary for the transcription of many genes involved in inflammatory and immune responses. In quiescent cells, NF-κB is held in the cytosol by an inhibitory protein, IkB. In stimulated cells, IkB is phosphorylated by IkB kinase (IKK) and targeted for proteasomal degradation, thus releasing the NF-κB dimer (most frequently composed of the p50 and p65 subunits), which moves to the nucleus and binds to DNA (reviewed in [5]). IKK is composed of two kinase subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma) [5]. ASA and salicylate inhibit the IKKbeta subunit of IKK kinase, thus preventing IkB phosphorylation and its subsequent degradation [6]. In endothelial cells, the salicylate inhibition of the TNF-α-induced expression of the adhesion molecules E-selectin, VCAM (vascular cell adhesion molecule), and ICAM (intracellular adhesion molecule) was ascribed to the inhibition of NF-κB signaling [7]. In monocytes, ASA inhibition of NF-κB activation was shown to down-regulate the production of IL-12 [8]. These findings seem to indicate that interference with the NF-κB transcriptional regulation may be an important component of the anti-inflammatory actions of salicylates. However, at similar concentrations, other effects of salicylates have also been reported. They were shown to interfere with signaling via the MAP kinases by inhibiting JNK and activating p38 in fibroblasts [9] and by inhibiting ERK, thus preventing integrin-dependent activation, in neutrophils [10]. The inhibition of leukocyte accumulation in vivo at the sites of carrageenan-induced inflammation was ascribed to a salicylate-induced production of adenosine [11].

Lymphocyte activation represents a central event in the inflammatory/immune response. In this study, we analyze the effects of salicylates on mitogen-induced lymphocyte proliferation. We show that proliferation is potently inhibited by theurapeutic concentrations of ASA and salicylate, but not by the COX inhibitor indomethacin. Furthermore, we show that proliferation is inhibited by a variety of agents that are known to inhibit NF-κB activation. We conclude that the mechanism by which salicylates inhibit lymphocyte proliferation is largely by inhibition of NF-κB signaling and that in general inhibition of NF-κB activation precludes lymphocyte proliferation.

Section snippets

Materials

RPMI (with l-glutamine), MTT, and PHA were from Sigma Chemical Co.; FBS, penicillin, streptomycin, and Lymphoprep™ from GIBCO BRL; oligonucleotide sense and antisense was synthesized by Amersham Pharmacia Biotech; poly(dI-dC), T4 polynucleotide kinase, and the radiochemicals [32P]ATP and [methyl-3H]thymidine were purchased from Amersham Pharmacia Biotech; parthenolide, MG-132, and SN-50 were from Biomol Research Laboratories, Bay 11-7082 from Calbiochem, and IL-2 from PeproTech EC LTD; human T

Results

Resting PBL were stimulated with the mitogen PHA. DNA synthesis was measured as [3H]thymidine incorporation after about 24 hr of stimulation. As shown in Fig. 1, thymidine incorporation was inhibited by ASA and salicylate. Semimaximal inhibition was obtained at approximately 1 mM of either agent, well within the concentrations (2–5 mM) required for the anti-inflammatory action of salicylate. The inhibition of PBL proliferation does not appear to depend on COX inhibition, first because

Discussion

In addition to its well-established role in activating the transcription of genes involved in immunological responses, studies indicate that NF-κB also functions in promoting cell proliferation. For instance, lymphocytes lacking the p50, p65, or c-Rel subunits of NF-κB exhibit defects in mitogenic responses [25], [26], [27], [28], and p50/p52 double-knockout animals fail to generate mature osteoclasts and B cells [29], [30]. Furthermore, deregulation of NF-κB activity has been associated with

Acknowledgements

This work was supported by a CNR strategic project contribution on apoptosis (No. 04960.ST74, 04899.ST74) and MURST grants 40 and 60%.

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    Abbreviations: ASA, acetylsalicylic acid; COX, cyclooxygenase; ERK, extracellular receptor-activated kinase; FBS, fetal bovine serum; IL-2, interleukin-2; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-KB, nuclear factor-kappa B; PBL, peripheral blood lymphocytes; PHA, phytohemagglutinin; and TNF-α, tumor necrosis factor alpha.

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