I am exploring regulation of inflammation in IBD, and looking closely at TH1 activations.

This includes how specific antigens and bacterial components may be recognized by macrophages and dendritic cells, and incite recruitment of monocytic stem cells into macrophages.

The monocytes possess chemokine receptors CCR2b, which are almost identical with angiotensin 2 receptors.

Some bacterial products and monocytic chemotactic protein 1(MCP-1) along with cysteinyl leukotrienes are ligands for these receptors, and evoke activation of IK B kinase thus allowing phosphorylation and activation of nuclear factor kappa beta (NFkappa beta). This then translocates to the cell nuclei where TNF alpha and IF gamma genes are activated to produce their products.

An important candidate gene for IBD is the NFKB1 gene located at chromosome 4q24. Nuclear Factor-B (NF-B) proteins are a family of transcription factors that regulate various biological defense processes, most notably innate and adaptive immune responses, acute phase reaction and apoptosis.

There are five members of the NF-B family in mammals: p50/p105, p65/RelA, c-Rel, RelB and p52/p100.

Although many dimeric forms of NF-B have been detected, the major form of NF-B is a heterodimer of the p50 and p65/RelA subunits, encoded by the genes NFKB1 and NFKB2, respectively). Human NFKB1 encodes two proteins, a 105 kDa, non DNA-binding, cytoplasmic molecule and a 50 kDa DNA-binding protein (p50) that corresponds to the N-terminus of p105. The NFKB1 gene spans 156 kb and has 24 exons with introns varying between 40 000 and 323 bp in length In most cells before stimulation, NF-B primarily resides in the cytoplasm in inactive complexes through association with a sequestering inhibitory protein, termed IB (12).

 A wide range of stimuli, including bacterial and viral products, cytokines and oxidant-free radicals, activate NF-B (9). These stimuli promote NF-B nuclear translocation by a mechanism that involves IB phosphorylation and the ubiquitin-proteosome pathway. This phosphorylation appears to target IB for degradation and leads to its dissociation from the NF-B complex and subsequent translocation of NF-B to the nucleus (13). There, active NF-B binds to genomic DNA at promoter regions and thereby regulates gene transcription.
 
TNF alpha induces the genes for inducible nitric oxide synthase called inducible nitric oxide synthase (INOS).  The nitric oxide which forms reacts with superoxide in mitochondria to produce the peroxynitrite, a more reactive free radical.  
 
Superoxides are also a product of oxidative injury in cells in circumstances of chemical and microbiological damage.   

The following abstracts address this matter.

Direct evidence of monocyte recruitment to inflammatory bowel disease mucosa.

Grimm MC, Pullman WE, Bennett GM, Sullivan PJ, Pavli P, Doe WF.

Division of Clinical Sciences, John Curtin School of Medical Research, Australian National University, Canberra, Australia.

Alterations in phenotype and function of intestinal macrophages occur in inflammatory bowel disease (IBD) but it is unclear whether these changes result from the recruitment of circulating monocytes to the intestine or from proliferation of resident intestinal macrophages. We sought to demonstrate the arrival of blood monocytes, the precursors of macrophages, in IBD mucosa.

Peripheral blood mononuclear cells were isolated from 23 patients with clinically active intestinal inflammation (13 Crohn's disease, eight ulcerative colitis, two infective colitis), then radiolabelled with 99mtechnetium (Tc)-stannous colloid (n = 13) or 111indium (In)-oxine (n = 10) before re-injection and abdominal scanning. Four patients had demonstrable intestinal monocyte uptake using [99mTc]-stannous colloid, while six [111In]-oxine-labelled monocyte scans were positive. Uptake sites correlated with actively inflamed regions. Patients demonstrating monocyte uptake had been treated with corticosteroids for a significantly (P < 0.02) shorter duration (median 3 vs 20 days) than those with negative scans. There was no significant difference between positive and negative scans for disease category, clinical or histological disease, activity, or radioisotope used. Biopsies of inflamed mucosa from two patients suffering ulcerative colitis who had positive scans showed a high proportion of CD14-positive macrophages, 4-9% of which contained autoradiographic grains. These results demonstrate that blood monocytes are recruited to the mucosa of actively inflamed bowel, and suggest that this process may be inhibited by corticosteroids. Moreover, the phenotype of the recently-arrived monocytes indicates their susceptibility to stimulation by lipopolysaccharide, and suggests a mechanism for the continuing inflammation in the bacterial product-rich milieu of IBD.

1: Clin Exp Allergy. 2005 Sep;35(9):1214-9.        

Cysteinyl leukotrienes induce monocyte chemoattractant protein 1 in human monocytes/macrophages.

Ichiyama T, Hasegawa M, Ueno Y, Makata H, Matsubara T, Furukawa S.

Department of Pediatrics, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan. ichiyama@yamaguchi-u.ac.jp

BACKGROUND: Monocytes/macrophages have a cysteinyl leukotriene 1 (CysLT1) receptor, but its function is poorly understood. Objective To elucidate the biological function of the CysLT1 receptor of human monocytes/macrophages. METHODS: We examined the production of TNF-alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and eotaxin induced by CysLTs (leukotriene (LT) C4, -D4, and -E4) in THP-1 cells, a human monocytic leukaemia cell line, and peripheral blood CD14+ monocytes/macrophages. Moreover, we examined the effect of CysLTs on the expression of beta-chemokine receptor 2B (CCR2B) as the receptor of MCP-1 by Western blot analysis.

RESULTS: ELISA revealed that CysLTs induced MCP-1 in THP-1 cells and peripheral blood CD14+ monocytes/macrophages, but not other cytokines. PCR demonstrated that CysLTs increased MCP-1 mRNA expression in THP-1 cells, and Western blotting showed that CysLTs increased the expression of CCR2B in THP-1 cells. Moreover, we demonstrated that pranlukast, a CysLT1 receptor antagonist, blocked MCP-1 production by CysLTs in THP-1 cells almost completely, and partially inhibited MCP-1 release by CysLTs in peripheral blood CD14+ monocytes/macrophages and CCR2B expression by CysLTs in THP-1 cells.

CONCLUSION: CysLTs induce MCP-1 and increase CCR2B expression in human monocytes/macrophages.

Nifedipine inhibited angiotensin II-induced monocyte chemoattractant protein 1 expression: involvement of inhibitor of nuclear factor kappa B kinase and nuclear factor kappa B-inducing kinase.

Wu L, Iwai M, Li Z, Li JM, Mogi M, Horiuchi M.

Department of Molecular and Cellular Biology, Division of Medical Biochemistry and Cardiovascular Biology, Ehime University School of Medicine, Shitsukawa, Tohon, Ehime 791-0295, Japan.

OBJECTIVE: The effect of nifedipine, a 1,4-dihydropyridine calcium antagonist, on the expression of monocyte chemoattractant protein 1 (MCP-1) induced by angiotensin II (Ang II) was examined using vascular smooth muscle cells (VSMC) isolated from rat thoracic aorta. METHODS AND RESULTS: Ang II increased the expression of MCP-1 messenger RNA accompanied by an increase in nuclear factor kappa B (NF-kappaB) binding activity to the cis DNA element in the promoter region of MCP-1. Ang II also decreased the cytosolic level of the inhibitor of NF-kappaB (IkappaB) and increased the phosphorylation of IkappaB subunits, IkappaBalpha and IkappaBbeta, as well as the phosphorylation of IkappaB kinase (IKK) subunits, IKKalpha and IKKbeta, suggesting that Ang II enhanced the breakdown of IkappaB. Nifedipine decreased MCP-1 mRNA expression, together with NF-kappaB binding activity to the promoter region of MCP-1 induced by Ang II. Nifedipine also attenuated the decrease in the cytosolic level of IkappaB, and the phosphorylation of IkappaB and IKK subunits induced by Ang II. Moreover, Ang II increased the phosphorylation of NF-kappaB-inducing kinase (NIK), and this increase was significantly inhibited by nifedipine.

CONCLUSION: As NIK is reported to activate IKK, our results suggest that nifedipine attenuates the effect of Ang II on MCP-1 expression in VSMC by regulating the activity of NF-kappaB through NIK, IKK and IkappaB.

  Putative Antibacterial Mechanisms for Angiotensin II Receptor Blockers

Authors: Trevor G Marshall, PhD1, Belinda Fenter, BS2, and Frances E Marshall, GradDipPharm, RPh3

email corresponding author: Trevor. m@yarcrip.com

telephone contact: +1-805-492-3693

Marshall TG, Fenter B, Marshall FE: Putative Antibacterial Mechanisms for Angiotensin II Receptor Blockers. JOIMR 2004;2(2):1

ABSTRACT

Angiotensin II has profound actions in Th1 immune disease. It directly modulates Nuclear Factor-kappaB (NF-kappaB), an essential precursor to the generation of inflammatory cytokines and chemokines, including TNF-alpha. Corticosteroids exert their anti-inflammatory action by totally shutting down activation of NF-kappaB, while Angiotensin Receptor Blockers (ARBs) inhibit excessive NF-kappaB activation, allowing the phagocytes to respond to immune challenge in a less aggressive manner. The two anti-inflammatory mechanisms are fundamentally different.

The new ARB, Olmesartan Medoxomil, has been identified as useful in treating Th1 inflammatory disease sarcoidosis, and the suggestion has been made that ARBs might also directly affect bacterial pathogens. Receptor proteins actively binding angiotensin II have been found on several species of bacteria. We found no match between the human angiotensin receptor and any similar protein in the 294 bacterial genomes currently sequenced, lending credence to the suggestion that bacteria may be using this host hormone in such a manner as to evade the host's immune system. Clearly, if an ARB was capable of actually inhibiting the supply of A-II, thus denying a microbe the ability to protect itself from destruction by phagocytosis, then that ARB could most definitely be classed as an 'antimicrobial'. The dosing of Olmesartan reported as most useful in immunomodulation, 40mg q8h, is well above the level needed to produce maximal hypotensive activity. It would thus seem likely that Olmesartan is acting on atypical receptors, perhaps directly upon pathogens, or upon some yet-to-be-defined human angiotensin-binding-protein(s).

Olmesartan fits into the binding pocket on the CCR2b receptor, and can block TH1 cytokine production. The effective dose appears to be about 40mg tds.

It is likely to be much safer than infliximab.

What we are seeing is recognition of other actions of known safe medications!

Lyprinol can markedly decrease leucotriene B4 production and this may be useful in Crohn’s disease.

Curcumin and Quercetin appear to have a protective effect.

Stinging nettle inhibits activation of NF kappa beta. So does curcumin in turmeric.

All of these agents may be useful to reduce doses of corticosteroid of pharmacological agents in current use. This is desirable if those agents have significant, medium or long term side effects.

New probiotics are also being assessed.

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