Lipoic acid downmodulates CD4 from human T lymphocytes by dissociation of p56Lck

doi:10.1016/j.bbrc.2006.03.172

Biochemical and Biophysical Research Communications

Volume 344, Issue 3, 9 June 2006, Pages 963-971

https://doi.org/10.1016/j.bbrc.2006.03.172 Get rights and content

Abstract

Lipoic acid is an antioxidant that suppresses and treats a model of multiple sclerosis, experimental autoimmune encephalomyelitis. We now demonstrate that treatment of human PBMC and T cell lines with LA downmodulated CD4 expression in a concentration-dependent manner. LA treatment of Con A stimulated PBMC specifically removed CD4 from the T-cell surface, but not CD3. Epitope masking by LA was excluded by using monoclonal antibodies targeting different domains of CD4. Incubation on ice inhibited CD4 removal following LA treatment, suggesting that endocytosis was involved in its downmodulation. LA is in a unique category of compounds that induce CD4 downmodulation by various mechanisms (e.g., gangliosides). We hypothesized that LA might induce dissociation of p56^Lck from CD4, thus leading to its downmodulation. Immunoblot analyses demonstrated reduced co-precipitation of p56^Lck from Jurkat T-cells following LA treatment and precipitation of CD4. This unique immunomodulatory effect of LA warrants further investigation.

Section snippets

Materials and methods

Reagents. LA was a gift from Integrative Technologies, Inc., (Wilsonville, OR). DHLA was generously provided by Dr. Rolf Winter at the Portland VA Medical Center (Portland, OR). Additional reagents used included: poly-d-Lysine (P-6407), protease inhibitor cocktail (P 8340), and Con A, which were obtained from Sigma; normal goat serum (NGS; 005-00-001), normal rabbit serum (NRS; 011-000-120), secondary antibodies: Rb anti-mouse HRP (315-035-003), Rb anti-goat HRP (305-035-003), Rb anti-mouse HRP

LA modulation of CD4 from PBMC and human T cell lines

Treatment of freshly isolated human PBMC with LA resulted in a concentration-dependent downmodulation of CD4 from the plasma membrane by LA (Fig. 1). DHLA, the reduced form of LA, treatment of PBMC did not induce downmodulation of CD4 at any concentration tested (Fig. 1). Two additional derivatives of LA, lipoamide (LPM; CONH₂ replaces the COOH of LA) and dimethyl lipoic acid (DMLA; SCH₃ replaces the sulfhydryl groups of DHLA) also had no effect on CD4 expression (data not shown).

Discussion

Herein we describe the novel observation of CD4 downmodulation by the antioxidant LA. Interestingly, its redox partner, DHLA, did not alter surface expression of CD4. Immunofluorescent analyses with monoclonal anti-CD4 antibodies recognizing different Ig-like domains within the CD4 protein support the conclusion that the CD4 is downmodulated by the LA, as opposed to epitope masking. Confocal microscopic analyses revealed the absence of CD4 from the plasma membrane and the cytosol, consistent

Acknowledgments

This work was supported by the Department of Veterans Affairs, National Institutes of Health AT P50 AT00066-01 and the Nancy Davis Center Without Walls. We wish to thank Drs. Rolf Winter and Michael Riscoe for the preparation of DHLA and Integrative Technologies, Inc. (Wilsonville, OR) for providing LA.

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Lipoic acid (LA), is an essential cofactor for mitochondria enzymes, with some biological functions, such as antioxidant and anti-inflammatory effects has been interested in diabetic neuropathy and neurodegenerative diseases. Multiple sclerosis (MS) is a chronic demyelinative inflammatory process of the central nervous system. Besides immunosuppressant drugs, nutritional supplement such as LA has been studied in MS. Pharmacokinetics (PK) evaluation found that LA had rapid absorption and clearance in human and animal models of MS. Furthermore, LA as an oral supplement was tolerated up to 1200 mg/days in MS patients and no severe side effects were reported. Generally, beneficial effects of LA in MS were observed in aspects of molecular mediators, antioxidant, and inflammatory markers. In conclusion, further trials with longer duration with clinical and imaging assessment are suggested.
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Whereas, histamine H2 receptors on peripheral monocytes stops interleukin (IL)-12 production (Elenkov et al., 1998), adenosine inhibits cytokine production in NK cells (Lokshin et al., 2006; Raskovalova et al., 2006) . Several researches are highlighting the effects of LA treatment, indeed the LA reduces or inhibits several immune processes that are involved in the inflammatory phenomena (Chaudhary et al., 2006; Kunt et al., 1999; Marracci et al., 2006; Salinthone et al., 2008). Following this line of thought, anti-inflammatory effects of LA may involve adenosine receptors (Salinthone et al., 2011).
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All treatments were performed independently at least three times. LA has been shown to exhibit anti-inflammatory properties in animal models of experimental autoimmune encephalomyelitis (EAE) and in human cells in vitro [4,6–8,28–31]. In an effort to determine the biochemical mechanisms that mediate the anti-inflammatory effects of LA, we discovered that LA stimulates cAMP production in immune cells [7,8].
Lipoic acid (LA) is a naturally occurring fatty acid that exhibits anti-oxidant and anti-inflammatory properties and is being pursued as a therapeutic for many diseases including multiple sclerosis, diabetic polyneuropathy and Alzheimer's disease. We previously reported on the novel finding that racemic LA (50:50 mixture of R-LA and S-LA) stimulates cAMP production, activates prostanoid EP2 and EP4 receptors and adenylyl cyclases (AC), and suppresses activation and cytotoxicity in NK cells. In this study, we present evidence that furthers our understanding of the mechanisms of action of LA. Using various LA derivatives, such as dihydrolipoic acid (DHLA), S,S-dimethyl lipoic acid (DMLA) and lipoamide (LPM), we discovered that only LA is capable of stimulating cAMP production in NK cells. Furthermore, there is no difference in cAMP production after stimulation with either R-LA, S-LA or racemic LA. Competition and synergistic studies indicate that LA may also activate AC independent of the EP2 and EP4 receptors. Pretreatment of PBMCs with KH7 (a specific peptide inhibitor of soluble AC) and the calcium inhibitor (Bapta) prior to LA treatment resulted in reduced cAMP levels, suggesting that soluble AC and calcium signaling mediate LA stimulation of cAMP production. In addition, pharmacological inhibitor studies demonstrate that LA also activates other G protein-coupled receptors, including histamine and adenosine but not the β-adrenergic receptors. These novel findings provide information to better understand the mechanisms of action of LA, which can help facilitate the use of LA as a therapeutic for various diseases.
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Alpha-lipoic acid (LA) has become a common ingredient in multivitamin formulas, anti-aging supplements, and even pet food. It is well-defined as a therapy for preventing diabetic polyneuropathies, and scavenges free radicals, chelates metals, and restores intracellular glutathione levels which otherwise decline with age. How do the biochemical properties of LA relate to its biological effects? Herein, we review the molecular mechanisms of LA discovered using cell and animal models, and the effects of LA on human subjects. Though LA has long been touted as an antioxidant, it has also been shown to improve glucose and ascorbate handling, increase eNOS activity, activate Phase II detoxification via the transcription factor Nrf2, and lower expression of MMP-9 and VCAM-1 through repression of NF-kappa B. LA and its reduced form, dihydrolipoic acid, may use their chemical properties as a redox couple to alter protein conformations by forming mixed disulfides. Beneficial effects are achieved with low micromolar levels of LA, suggesting that some of its therapeutic potential extends beyond the strict definition of an antioxidant. Current trials are investigating whether these beneficial properties of LA make it an appropriate treatment not just for diabetes, but also for the prevention of vascular disease, hypertension, and inflammation.
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Published by Elsevier Inc.

Lipoic acid downmodulates CD4 from human T lymphocytes by dissociation of p56Lck

Abstract

Section snippets

Materials and methods

LA modulation of CD4 from PBMC and human T cell lines

Discussion

Acknowledgments

J. Autoimmun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Virology

Gen. Pharmacol.

Biochem. Pharmacol.

J. Neuroimmunol.

Immunopharmacology

Free Radic. Biol. Med.

Mol. Immunol.

J. Neuroimmunol.

Immunological aspects of demyelinating diseases

Annu. Rev. Immunol.

Axonal transection in the lesions of multiple sclerosis

N. Engl. J. Med.

Candidate autoantigens in multiple sclerosis

Mult. Scler.

Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis

J. Immunol.

T cells specific for the myelin oligodendrocyte glycoprotein mediate an unusual autoimmune inflammatory response in the central nervous system

Eur. J. Immunol.

Identification of autoantibodies associated with myelin damage in multiple sclerosis

Nat. Med.

Experimental allergic encephalomyelitis (EAE) in mice lacking CD4+ T cells

Eur. J. Immunol.

CD4 and the immunoglobulin superfamily

Philos. Trans. R. Soc. Lond. B. Biol. Sci.

Involvement of both major histocompatibility complex class II alpha and beta chains in CD4 function indicates a role for ordered oligomerization in T cell activation

J. Exp. Med.

Lipoic acid downmodulates CD4 from human T lymphocytes by dissociation of p56^Lck

Experimental allergic encephalomyelitis (EAE) in mice lacking CD4⁺ T cells