The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.

Objectives Apolipoprotein E (apoE) exerts potent anti-inflammatory effects. We here investigated the effect of apoE on the functional phenotype of macrophages. Methods and Results Human apoE receptors VLDL-R or apoER2 were stably expressed in RAW264.7 mouse macrophages. In these cells apoE downregulated markers of the pro-inflammatory M1 phenotype (iNOS, IL-12, MIP-1α), but upregulated markers of the anti-inflammatory M2 phenotype (arginase-I, SOCS3, IL-1RA). In addition, M1 macrophage responses (migration, generation of reactive oxygen species, antibody-dependent cell cytotoxicity, phagocytosis) as well as poly(I:C)- and/or IFN-γ-induced production of pro-inflammatory cytokines, COX-2 expression, and activation of NF-κB, IκB and STAT1 were suppressed in VLDL-R- or apoER2-expressing cells. Conversely, the suppression of M2 phenotype and the enhanced response to poly(I:C) were observed in apoE-producing bone marrow macrophages derived from VLDL-R-deficient mice, but not wild type or LDL receptor-deficient mice. The modulatory effects of apoE on macrophage polarization were inhibited in apoE receptor-expressing RAW264.7 cells exposed to SB220025, a p38MAP kinase inhibitor, and PP1, a tyrosine kinase inhibitor. Accordingly, apoE induced tyrosine kinase-dependent activation of p38MAP kinase in VLDL-R- or apoER2-expressing macrophages. Under in vivo conditions, apoE−/− mice transplanted with apoE-producing wild-type bone marrow showed increased plasma IL-1RA levels and peritoneal macrophages of transplanted animals were shifted to the M2 phenotype (increased IL-1RA production and CD206 expression). Conclusion ApoE signaling via VLDL-R or apoER2 promotes macrophage conversion from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. This effect may represent a novel anti-inflammatory activity of apoE.

The ␤ 2 integrins are important for transendothelial migration of leukocytes as well as for T-cell activation during antigen presentation. Despite abundant expression of ␤ 2 integrins on antigen-presenting cells (APCs), their functional relevance for antigen presentation is completely unclear. We show here that dendritic cells (DCs) from CD18-deficient mice, which lack all functional ␤ 2 integrins, have no defect in antigen presentation. Moreover, DCs from normal mice express inactive ␤ 2 integrins that do not become activated on contact with T cells, at least in vitro. Pharmacologic activation of ␤ 2 integrins on DCs results in a significant reduction of their T cell-activating capacity. This effect is mediated by Mac-1 (CD11b/CD18) on DCs because it could be reversed via blocking antibodies against CD18 and CD11b. Furthermore, the antigen-presenting capacity of macrophages, which express constitutively active ␤ 2 integrins, is significantly enhanced on Mac-1 blockade. We therefore conclude that active CD11b/ CD18 ( IntroductionThe ␤ 2 integrins (CD11/CD18) are heterodimeric leukocyte adhesion molecules exclusively expressed on hematopoietic cells. They play an important role for cell-to-cell contacts between leukocytes as well as for contacts between leukocytes and endothelial cells. 1,2 The common ␤ chain (CD18) associates with 4 different ␣ subunits, ␣L, ␣M, ␣X, and ␣D, forming distinct functional heterodimers termed leukocyte functional antigen-1 (LFA-1, CD11a/CD18), Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18), and CD11d/CD18. [2][3][4] These interact with more than 20 ligands, many of which belong to the family of ICAMs. 5,6 Adhesion between T cells and antigen-presenting cells (APCs) is necessary for the formation of the immunologic synapse (IS). 7,8 Interactions between LFA-1 on the surface of T cells and intercellular adhesion molecules ICAM-1, 2, and 3 (in humans) on the APC surface were described to be involved in formation of the IS. 9,10 Moreover, ligation of LFA-1 on T cells is required for optimal activation and differentiation of T cells, [11][12][13][14][15][16][17][18][19][20][21] although the downstream signaling events are still not fully understood. On T cells, experiments with blocking antibodies against LFA-1 and with LFA-1-deficient T cells have shown that those T cells are impaired in numerous effector functions. [22][23][24] Dendritic cells (DCs), on the other hand, are known to be the most relevant APCs in the immune system. 25,26 DCs express at least 3 ␤ 2 integrins, LFA-1 (CD11a/CD18), Mac-1 (CD11b/ CD18), and gp150,95 (CD11c/CD18). 27,28 Compared to LFA-1 on T cells, very little information is available about the role and function of ␤ 2 integrins on DCs, especially regarding the process of antigen presentation and T-cell activation. Studies with splenocytes of mice deficient in CD11a, CD11b, CD11c, and CD11d showed impaired T-cell activation in CD11a-, CD11b-, and CD11d-deficient splenocytes on stimulation with superantigen, but this was due to dysfunctional T cells rather than a de...

Glucocorticoids (GC) are still the most widely used immunosuppressive agents in clinical medicine. Surprisingly, little is known about the mechanisms of GC action on monocytes, although these cells exert pro- and anti-inflammatory effects. We have shown recently that GC induce a specific monocyte phenotype with anti-inflammatory properties in humans. We now investigated whether this also applies for the murine system and how this subset would relate to recently defined murine subtypes. After treatment with dexamethasone for 48 h, monocytes up-regulated scavenger receptor CD163 and Gr-1, down-regulated CX(3)CR1, and shared with human GC-treated monocytes functional features such as low adhesiveness but high migratory capacity. They specifically up-regulated anti-inflammatory IL-10, but not TGF-beta, and in contrast to their human counterparts, they down-regulated IL-6. Although GC-induced monocytes down-regulated CX(3)CR1, a distinctive marker for classical/proinflammatory human and murine monocytes (CX(3)CR1(lo)CCR2(+)Ly6C(hi)), they differed from this physiologically occurring subset, as they remained Ly6C(med) and unactivated (CD62 ligand(++)). In addition to their immunosuppressive effects, they were CD11b(+)Gr-1(+) and expressed the IL-4Ralpha chain (CD124), a recently described, signature molecule of tumor-induced myeloid-derived suppressor cells (MDSC). We therefore generated murine MDSC in B16 melanoma-bearing mice and indeed found parallel up-regulation of CD11b(+)Gr-1(+) and CD124 on GC-induced monocytes and MDSC. These data allow us to speculate that the GC-induced subtype shares with inflammatory monocytes the ability to migrate quickly into inflamed tissue, where they, however, exert anti-inflammatory effects and that similarities between GC-induced monocytes and MDSC may be involved in progression of some tumors observed in patients chronically treated with GC.

Human S100A12 is an endogenous TLR4 ligand that induces monocyte activation, thereby acting as an amplifier of innate immunity during early inflammation and the development of sepsis.

IntroductionFor activation, T cells must physically engage antigen-presenting cells (APCs). 1 With B cells as APCs, the interaction plane, termed "immunologic synapse," 2 is a highly organized structure of signaling and adhesion molecules. 3 A mature synapse requires at least 30 minutes of stable cell-cell interaction to form 2 and can last for several hours in vitro 4 and in explanted lymph nodes. 5 Although investigated mostly with B cells or surrogate APCs in liquid cultures, the concept of long-term contacts has previously been considered a universal feature of all cognate T cell-APC (T-APC) interactions. 6 However, in promigratory collagen matrices as environment for T-APC interaction also dynamic and short-lived encounters to dendritic cells (DCs) are able to induce the full spectrum of T-cell signaling and activation. 7 Here, T cells engage DCs only for a few minutes, detach, and establish new contacts to neighboring DCs until activation is achieved. 8 Migrating T cells can drag a mature synapse over the surface of an APC, showing the principal compatibility of T-cell migration and synapse maintenance. 9 Furthermore, studies on APC-T interactions in explanted lymph nodes 10 or thymic organ cultures 11 have observed both static and dynamic interactions between T cells or thymocytes and local APCs. Intravital imaging revealed a 3-phase model of T-APC interaction in a lymph node, in which CD8 T cells displayed a period of 8 hours, characterized by short-lived and dynamic DC scanning, followed by 12 hours with stable interactions to single DCs and a return to high motility afterward. 12 During adhesive contacts, T cells maintained considerable cytoskeletal activity and shape change. 12 Weaver's group (Hurez et al 13 and Saparov et al 14 ) described a heterogeneity in naive T cells, leading to a majority of short-interacting cells, which could proliferate but did not produce interleukin 2 (IL-2), the latter only being produced by T cells making long contacts to APCs. T-cell receptor (TCR)-proximal signals such as phosphorylation of zeta-associated protein 70 (ZAP-70) or Lck 15 are only detectable for the first 15 minutes after the onset of a contact and are already absent before a synapse has fully matured. 16 In contrast, active phosphatidyl inositol 3 (PI3)-kinase is located in an initial segment (IS) over the entire period of T-APC interaction and interference with this process inhibits T-cell activation. 17,18 However, besides a continuous TCR signal, also discontinuous signaling can lead to T-cell activation and effector function. 19 Together, these observations suggest that a spectrum of interaction modes, reaching from static adhesion and clustering to dynamic migratory encounters with APCs, can lead to the activation of T cells. 8,20 So far no systematic analysis on the biophysical and molecular principles underlying static versus dynamic interactions has been performed. According to one hypothesis, the nature of the APC, which is encountered by the T cell, is decisive for the mode of interaction, 8 wh...

CD40–CD40 ligand (L) interactions play a pivotal role in immune-mediated inflammatory responses via the activation of antigen-presenting cells (APCs). To investigate the effects of continuous activation of resident tissue APCs, in this case the Langerhans cells (LCs) of the skin, CD40L expression was targeted to the basal keratinocytes of the epidermis of mice using the keratin-14 promoter. Approximately 80% of the transgenic (Tg) mice spontaneously developed dermatitis on the ears, face, tail, and/or paws. Compared with littermates, Tgs had a >90% decrease in epidermal LCs yet increased numbers within the dermis suggestive of enhanced emigration of CD40-activated LCs. Tgs also displayed massive regional lymphadenopathy with increased numbers of dendritic cells and B cells. Moreover, a decrease in IgM and an increase in IgG1/IgG2a/IgG2b/IgE serum concentrations was detectable. Screening for autoantibodies revealed the presence of antinuclear antibodies and anti-dsDNA antibodies implicative of systemic autoimmunity. Accordingly, renal Ig deposits, proteinuria, and lung fibrosis were observed. Adoptive transfer of T cells from Tgs to nonTg recipients evoked the development of skin lesions similar to those found in the Tgs. Dermatitis also developed in B cell–deficient CD40L Tg mice. These findings suggest that in situ activation of LCs by CD40L in the skin not only leads to chronic inflammatory dermatitis but also to systemic mixed-connective-tissue-like autoimmune disorders, possibly by breaking immune tolerance against the skin.

Proteinase-activated receptor-2 (PAR2) belongs to a new G protein-coupled receptor subfamily activated by serine proteinases. PAR2 has been demonstrated to play a role during inflammation and immune response in different tissues including the skin. We examined whether PAR2 is functionally expressed by cutaneous human primary skin mast cells (HPMC) and the human mast cell line 1 (HMC-1). Reverse transcription-polymerase chain reaction and FACS analysis show expression of PAR2 both at the RNA and protein level. HPMCs and HMC-1 also express PAR1, PAR3, and PAR4. Ca-mobilization studies demonstrate functional PAR2 expressed by human skin mast cells, as shown by natural and synthetic PAR2 agonists. PAR2 agonists induced histamine release from HPMC indicating a role of PAR2 in regulating inflammatory and immune responses by skin mast cells. Double-immunofluorescence staining reveals colocalization of PAR2 with tryptase in the majority of human skin mast cells. In conclusion, trypsin and tryptase as well as specific agonists for PAR2 were able to induce Ca2+ mobilization in HPMCs, and agonists of PAR2 induce the release of histamine from these cells. Thus, PAR2 may be an important regulator of skin mast cell function during cutaneous inflammation and hypersensitivity.