Lipopolysaccharide (LPS) from Gram-negative bacteria is one of the most potent innate immune-activating stimuli known. Here we review the current understanding of LPS effects on human monocyte and macrophage function. We provide an overview of LPS signal transduction with attention given to receptor cooperativity and species differences in LPS responses, as well as the role of tyrosine phosphorylation and lysine acetylation in signalling. We also review LPS-regulated transcription, with emphasis on chromatin remodeling and primary versus secondary transcriptional control mechanisms. Finally, we review the regulation and function of LPS-inducible cytokines produced by human monocytes and macrophages including TNFα, the IL-1 family, IL-6, IL-8, the IL-10 family, the IL-12 family, IL-15 and TGFβ.

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To detect mRNA expression of nitric oxide synthase (NOS) isoforms in human monocytes/macrophages reverse transcription polymerase chain reaction (RT-PCR) was used. mRNA was isolated from stimulated or unstimulated monocytes/macrophages and RT-PCR was performed using oligonucleotide primers derived from mRNA sequences of either human endothelial constitutive (c) or human hepatocyte inducible (i) NOS. RT-PCR of mRNA isolated from resting monocytes and macrophages resulted in the amplification of a cNOS specific mRNA fragment. When the cells were stimulated with lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) prior to mRNA extraction, RT-PCR yielded an iNOS-specific amplification product. Whereas the activation of both cell types was accompanied by expression of iNOS mRNA, the cNOS signal seemed to be diminished upon immunostimulation. Not only in purified human monocytes but also in the human monocytoid cell lines MonoMac 6, THP-1, and U937 cNOS mRNA was detected. The data clearly demonstrate the presence of iNOS and cNOS mRNA in human monocytes/macrophages and provide the necessary tools to investigate the regulation of NO synthesis in these cell populations.

Monocytes and monocytic cells produce proinflammatory cytokines upon direct cell contact with activated T cells. In the autoimmune disease rheumatoid arthritis, the pivotal role of TNF-α implies that the interaction between transmembrane TNF-α (mTNF) and the TNF receptors (TNFR1 and TNFR2) might participate in the T cell contact-dependent activation of monocytes. Accordingly, treatment of rheumatoid arthritis by administration of a TNF-α-blocking Ab was found to significantly decrease TNF-α production by monocytes. Several lines of evidence indicated that signaling through TNFR1/2 and through mTNF (reverse signaling) is involved in TNF-α production by monocytes after T cell contact: 1) blocking mTNF on activated T cells leads to a significant reduction in TNF-α production; 2) down-regulation of TNFR1/2 on monocytes by transfection with small interfering RNA results in diminished TNF-α production; 3) blocking or down-regulating TNFR2 on activated T cells inhibits TNF-α production, indicating that mTNF on the monocyte surface mediates signaling; 4) ligation of mTNF on monocytes by surface TNFR2 transfected into resting T cells induces TNF-α production due to reverse signaling by mTNF; and 5) ligation of mTNF on monocytes by a soluble TNFR2:Ig receptor construct induces TNF-α production due to reverse signaling. In conclusion, we identified mTNF and TNFR1/2 as interaction partners contributing to TNF-α production in monocytes. Both pathways initiated by mTNF-TNFR interaction are likely to be inhibited by treatment with anti-TNF-α Abs.

We investigated whether sodium diethyldithiocarbamate @ETC), an inhibitor of the nuclear transcription factor kappa B (NFkappaB), modulates induction of NO synthase (NOS) in murine bone marrow-derived macrophages. A short exposure (between 1 and 16 h) of L929-cell mediumpreconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1-l mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappaB is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of the gene encoding inducible NOS.NO synthase; Transcription; NFkappaB; Diethyldithiocarbamate; Bone marrow-derived macrophage 1, INTRODUCTION Intra-and extracellular pathogens, their breakdown products, several cytokines and a plethora of other noxious agents and conditions induce the expression of NO synthase in mammalian cells (for review see [l-3]). NO generated from L-arginine by the inducible NO synthase (iNOS) is regarded as a defense effector molecule with cytotoxic/cytostatic and microbicidal/microbiostatic activity [1,2]. To date little or nothing is known about the transduction cascade that unifies all these pathogenic signals translating them into activation of the iNOS gene. Eukaryotic gene expression is controlled by promotor and enhancer DNA sequences, which are activated by specific protein factors. One of the nuclear transcription factors initially identified in B-cells and monocytes, the multiprotein complex NFkappaB [4], is rapidly activated upon perturbation of cells by conditions similar to those known to induce NOS. NFkappaB is known to mediate the immediate-early gene response by enhancing the transcription of a multitude of genes encoding defense and signalling proteins, such as cytokines and cytokine receptors [4]. Activation of NFkappaB can be specifically prevented by antioxidants, thiols and iron chelators, and especially by dithiocarbamates [4]. To clarify whether NFkappaB participates in the induction of NOS, we assessed the effect of diethyldithiocarbamate (DETC) on iNOS mRNA levels and expression of NOS activity in lipopolysaccharide (LPS)-stimulated murine bone marrow-derived macrophages. MATERIALS AND METHODSBone marrow cells from the femurs of Balb/c mice were isolated and cultured in L929-cell conditioned medium as described recently [5]. Cells grown on culture dishes (3.5 cm diameter; 10' cells/dish) were exposed to LPS (0.1 &ml; E. coli serotype 055:B5), DETC (0.01 to 1 mM; both from Sigma, Deisenhofen, Germany) and hemin (Normosang, Leiras, Turku, Finland) according to the protocols described in section 3.Messenger RNA encoding iNOS was assessed by Northern blot technique using a HincIIlSSP I fragment of the mouse iNOS [6] as a cDNA probe. The cDNA probe was a generous gift of Drs. Q. Xie and C.F. Nathan, Cornell University Medi...

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable nonselective cation channel that is stimulated by oxidative stress and specifically activated by intracellular ADP-ribose. Because TRPM2 is highly expressed in immunocytes, a role of this channel in inflammation processes has been proposed. The aim of the current study was to determine the function of TRPM2 in LPS-induced cytokine production of human monocytes. Incubation of human primary monocytes with LPS resulted in an upregulation of TRPM2 mRNA, protein, and of ADP-ribose–induced membrane currents. By using short hairpin RNA to downregulate TRPM2 expression in THP-1 monocytes, we demonstrate that TRPM2 is required for the LPS-induced production of IL-6, IL-8, IL-10, and TNF-α. Application of LPS led to a time-dependent increase in intracellular Ca2+ concentrations in THP-1 cells that was clearly reduced by downregulation of TRPM2. Omission of extracellular Ca2+ strongly decreased TNF-α production in TRPM2-expressing cells. Thus, TRPM2-mediated Ca2+ entry is a central mechanism for LPS-induced cytokine production in monocytic cells. The identification of TRPM2 as a major player in this LPS-dependent process makes it a promising tool in modulating monocyte functions.

SUMMARYMonocytes (MO) and macrophages (MAC ) are important producers of cytokines involved in the pathophysiology of bacterial sepsis. Most studies concentrate on the effects of bacterial lipopolysaccharides (LPS) regarding the induction of cytokine gene expression and secretion in MO/MAC. Here we report that besides LPS, the synthetic lipoprotein analogue lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyl )-(2RS)-propyl )-(R)-cysteinyl-alanyl-glycine (Pam3-Cys-Ala-Gly), another component of the outer membrane of Gram-negative bacteria, as well as heat-killed Staphyloccocus aureus (S. aureus/SAC ) are potent stimuli for cytokines in human MO. For all three investigated stimuli we found an individual pattern of cytokine induction: LPS was most potent in inducing interleukin-6 (IL-6) synthesis, whereas for tumour necrosis factor-a ( TNF-a) secretion SAC was the best stimulus. Comparable amounts of IL-8 were induced by either LPS or Pam3-Cys-AlaGly, with SAC being less effective even at higher concentrations. The addition of serum led to an increase in LPS-, SAC-and Pam3-Cys-Ala-Gly-stimulated TNF-a secretion, indicating that the presence of serum is critical not just for LPS stimulation. Furthermore, as is known for LPS, Pam3-Cys-Ala-Gly and SAC rendered MO refractory to a second bacterial stimulus. Pam3-CysAla-Gly and SAC induced tolerance for itself, but LPS could partially overcome this effect. As the CD14 molecule is discussed as a common receptor for different bacterial components, we investigated whether the TNF-a response of MO could be blocked by anti-CD14 antibodies. MY4, a CD14 antibody, selectively blocked the TNF-a secretion induced by LPS but not by Pam3-Cys-Ala-Gly or SAC. In summary, we conclude that besides LPS, lipopeptide Pam3-CysAla-Gly and SAC are potent stimuli for human MO, while the mechanisms of activation seem to be partially different from LPS.

Colloidal Force Microscopy was employed to study the viscoelastic and adhesive properties of macrophages upon stimulation with lipopolysaccharide (LPS). Force vs. distance measurements were performed. The adhesion of LPS-stimulated cells (separation force = 37 ± 3 nN) was almost twice as high as that of resting macrophages (16 ± 1 nN). Upon retraction pulling of membrane tethers was observed. Tether lengths and forces at which rupture take place did not depend on stimulation. The reduced Young's modulus K, a measure of cytoskeleton elasticity, was three times lower than that of the control. The data show that LPS has profound effects on cytomechanical and adhesion properties of macrophages.