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The Keratinocyte as a Target for Staphylococcal Bacterial Toxins

  • Jeffrey B. Travers
    Correspondence
    The H.B Wells Center for Pediatric Research, Riley Hospital for Children Room 2659, 635 Barnhill Drive, Indianapolis, Indiana 46202
    Affiliations
    Departments of Dermatology, Pediatrics, Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A.
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  • David A. Norris
    Affiliations
    Department of Dermatology, University of Colorado School of Medicine, and Department of Veterans Affairs Hospital, Denver, Colorado, U.S.A.
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  • Donald Y.M. Leung
    Affiliations
    Departments of Pediatrics, The National Jewish Medical and Research Center, and The University of Colorado School of Medicine, Denver, Colorado, U.S.A.
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      Skin infections with Staphylococcus aureus are not only an important cause of morbidity and even mortality, but are thought to serve as initiation and/or persistance factors for numerous inflammatory skin diseases, including psoriasis and atopic dermatitis. One mechanism by which S. aureus can modulate the immune system is through the production of proteins such as superantigenic toxins, Protein A, as well through the cytolytic α-toxin. This review serves to discuss the biology of these three types of proteins, with emphasis on their ability to stimulate the production of powerful pro-inflammatory lipid- and protein-derived cytokines in keratinocytes. Characterization of interactions between these proteins and the keratinocyte can provide a better understanding of how bacterial infection modulates inflammatory skin diseases, as well as provide the basis for improved therapies involving antibacterial agents.

      Keywords

      Abbreviations

      CLA
      cutaneous lymphocyte-associated antigen
      MHC II
      major histocompatibility class II protein
      PAF
      platelet-activating factor
      PG
      prostaglandin
      SAg
      superantigen
      TCR
      T cell receptor
      Skin infections with Staphylococcus aureus are an important cause of morbidity and even mortality. Infections by these bacteria not only cause disease directly, but are thought to be important triggers in worsening inflammatory skin diseases ranging from atopic dermatitis to psoriasis (reviewed by
      • Leung D.Y.M.
      • Hauk P.
      • Strickland I.
      • Travers J.B.
      • Norris D.A.
      The role of superantigens in human diseases: therapeutic implications for the treatment of skin diseases.
      ). One mechanism by which S. aureus can accomplish this is through the production of soluble proteins with potent immunomodulatory effects. The objective of this review is to focus on one aspect of cutaneous S. aureus bacterial infection; how interactions between keratinocytes and bacterial proteins such as superantigens, α-toxin, and Protein A can potentially affect the inflammatory process.

      The keratinocyte as an active participant in cutaneous inflammation

      Keratinocytes are by far the most abundant cell type in the epidermis, and our concept of the keratinocyte's role in inflammatory processes has changed greatly from a passive target to an active participant. The keratinocyte has the ability to produce and release numerous lipid and protein cytokines (reviewed by
      • Greaves M.W.
      • Camp R.D.R.
      Prostaglandins leukotrienes, phospholipase, platelet-activating factor and cytokines: an integrated approach to inflammation of human skin.
      ;
      • Kupper T.S.
      Immune and inflammatory processes in cutaneosu tissues: mechanisms and speculations.
      ). Keratinocytes also can express adhesion molecules that allow enhanced leukocyte interactions. These properties, as well as the relative long life of a keratinocyte compared with other inflammatory cells such as the neutrophil or eosinophil, make the keratinocyte a potentially important player in the cutaneous inflammatory response.

      Keratinocyte-derived cytokines

      Keratinocytes have the capacity to synthesize and release a wide variety of immunomodulatory molecules (a partial list is found in Table I). Keratinocyte-derived pro-inflammatory and immunomodulatory molecules can be divided into those that are lipid versus those that are protein based. Lipid-derived inflammatory mediators produced by keratinocytes include derivatives of phospholipids [platelet-activating factor (1-O-alkyl-2-acetyl glycerophosphocholine; PAF) and lyso-phosphatidic acid (LPA)], arachidonic acid metabolites (eicosanoids), and others including ceramides. These mediators tend to be synthesized by keratinocytes in response to membrane damage. Of significance, essentially all of these lipid-derived mediators are produced in response to an increased calcium mobilization signal, which can be seen following membrane damage.
      Table IImmunomodulatory molecules produced by keratinocytes
      Lipid-derivedProtein-derived
      Prostaglandins E2, D2, F2-αInterleukins 1, 6, 8, 12, 15, 18
      12-Hydroxyeicosatetraenoic acidTumor necrosis factor-α
      Platelet-activating factorTransforming growth factor-α
      Lyso-phosphatidic acidInterferon- β
      CeramidesGranulocyte and Granulocyte macrophage colony stimulatory factors
      The major eicosanoid synthesized by human keratinocytes is PGE2, with lesser amounts of PGD2 and PGF2-α (
      • Pentland A.P.
      • Needleman P.
      Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis.
      ). Injection of nanogram amounts of PGE2 causes erythema in human skin (
      • Camp R.D.R.
      • Greaves M.W.
      Inflammatory mediators in the skin.
      ). Larger amounts induce swelling through vasodilatory effects on blood vessels, with only minimal leukocyte chemoattractive properties. PGD2 also has erythrogenic effects, as well as granulocyte chemoattractant properties (
      • Woodward D.F.
      • Hawley S.B.
      • Williams L.S.
      • et al.
      Studies on the ocular pathology of PGD2.
      ). Though keratinocytes synthesize hydroxyeicosateranoic acids (HETE), they do not synthesize significant levels of leukotrienes (
      • Greaves M.W.
      • Camp R.D.R.
      Prostaglandins leukotrienes, phospholipase, platelet-activating factor and cytokines: an integrated approach to inflammation of human skin.
      ).
      Produced by the subsequent actions of phospholipase A2 and PAF acetyltransferase, PAF is synthesized in response to the same stimuli that induce eicosanoid production (
      • Pinckard R.N.
      • Woodard D.S.
      • Showell H.J.
      • et al.
      Structural and (patho) physiological activity of platelet-activating factor.
      ). Intradermal injection of PAF results in a painful wheal and flare reaction within minutes. Human keratinocytes both synthesize PAF as well as express functional PAF receptors (PAF-R) (
      • Michel L.
      • Denizot Y.
      • Thomas Y.
      • et al.
      Production of PAF-acether by human epidermal cells.
      ;
      • Travers J.B.
      • Huff J.C.
      • Rola-Pleszcynski M.
      • Gelfand E.W.
      • Morelli J.G.
      • Murphy R.C.
      Identification of functional paltelet-activating factor receptors on human keratinocytes.
      ). Activation of the epidermal PAF-R results in the biosynthesis of numerous mediators, including eicosanoids, TNF-α, IL-6, IL-8, and PAF itself (
      • Pei Y.
      • Barber L.A.
      • Murphy R.C.
      • et al.
      Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.
      ).
      Amongst the protein-derived cytokines produced by keratinocytes, TNF-α and IL-1 have been termed primary cytokines due to their ability to induce inflammatory reactions in part through the ability to induce a host of other lipid and protein mediators (
      • Kupper T.S.
      Immune and inflammatory processes in cutaneosu tissues: mechanisms and speculations.
      ). TNF-α and IL-1 both exert effects on receptors expressed on keratinocytes that are linked to the nuclear factor >B pathway, with resultant production of cascades of other protein and lipid cytokines. IL-6 is a pleiotropic cytokine with effects on both B and T cells (
      • Paquet P.
      • Pierard G.E.
      Interleukin-6 and the skin.
      ). IL-7 has the ability to stimulate the growth of both progenitors and mature B and T cells (
      • Heufler C.
      • Topar G.
      • Grasseger A.
      • et al.
      Interleukin 7 is produced by murine and human keratinocytes.
      ). IL-8 is a potent neutrophil chemoattractant also produced by keratinocytes. IL-12 is a potent TH1 cytokine that promotes activation of macrophages that can be synthesized by keratinocytes in response to varied stimuli including phorbol esters and IFN-γ (
      • Aragane Y.
      • Riemann H.
      • Bhardwaj R.S.
      • et al.
      Interleukin-12 is expressed and released by human keratinocytes and epidermoid carcinoma cell lines.
      ). In addition, bacterial superantigens appear to induce production of skin-homing (cutaneous lymphocyte-associated antigen-positive; CLA) T cells through an IL-12-dependent process (
      • Leung D.Y.M.
      • Travers J.B.
      • Giorno R.
      • et al.
      Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.
      ). Though resting keratinocytes do not synthesize the T cell activating cytokine IL-15, its production can be induced by IFN-γ, a process inhibited by dexamethasone (
      • Han G.W.
      • Iwatsuki K.
      • Inoue M.
      • et al.
      Interleukin-15 is not a constituitive cytokine in the epidermis, but is inducible in culture or inflammatory conditions.
      ). IL-18, a novel cytokine that strongly induces IFN-γ production in T cells, has also been shown to be synthesized by human and murine keratinocytes. Hyperosmolar stress induced by mannitol or NaCl is a potent stimulus for IL-18 production in both epithelial and endothelial cells, suggesting that this cytokine could be important in damage-mediated inflammatory responses (
      • Takeuchi M.
      • Okura T.
      • Mori T.
      • et al.
      Intracellular production of interleukin-18 in human epithelial cell lines is enhanced by hyperosmotic stress in vitro.
      ). Thus, a keratinocyte that sustains membrane damage has the potential to synthesize and release numerous lipid and protein cytokines that could modulate the inflammatory response.

      Keratinocyte adhesion molecules

      In addition to the ability of keratinocytes to modulate inflammation through soluble mediators, these cells can also express membrane-associated molecules that can serve to bind immune cells as well as to stimulate signal transduction pathways leading to further production of mediators. Through its ability to bind the B-2 integrin leukocyte function-associated antigen found on most leukocytes, the adhesion molecule intracellular adhesion molecule-1 (ICAM-1; CD54) is an important mediator of cellular immune reactions (reviewed by
      • Krutmann J.
      • Grewe M.
      Involvement of cytokines, DNA damage, and reactive oxygen intermediates in ultraviolet radiation-induced modulation of intracellular adhesion molecule-1 expression.
      ). Though not found on resting epidermal cells, ICAM-1 surface expression can be induced by cytokines including TNF-α (
      • Krutmann J.
      • Kock A.
      • Schauer E.
      • et al.
      Tumor necrosis factor and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression.
      ) and PAF (
      • Pei Y.
      • Dy L.C.
      • Natarajan S.
      • Travers J.B.
      Activation of the epidermal platelet-activating factor receptor results in ICAM-1 expression.
      ), as well as by physical methods including tape-stripping (
      • Nickoloff B.J.
      • Naidu Y.
      Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin.
      ). Similar to ICAM-1, resting keratinocytes do not express the costimulatory molecule B7-1 (CD80), yet epidermal expression can be seen during allergic and contact dermatitis (
      • Wakem P.
      • Burns Jr., R.P.
      • Ramirez F.
      • et al.
      Allergens and irritants transcriptionally upregulate CD80 gene expression in human keratinocytes.
      ). The significance of epidermal B7-1 expression is not clear, though this signaling molecule usually associated with antigen-presenting cells prevents the induction of T cell anergy and induces IL-2 production during antigen presentation. Of note, transgenic mice overexpressing B7-1 on keratinocytes exhibit exaggerated and persistent contact hypersensitivity reactions to a variety of haptens (
      • Gaspari A.A.
      • Burns Jr., R.P.
      • Kondo S.
      • et al.
      Characterization of the altered reactivity of transgenic mice whose keratinocytes overexpress B7–1.
      ).
      In addition to adhesion and costimulatory molecules, keratinocytes can also express MHC-II molecules (i.e., HLA-DR) in response to various pro-inflammatory and immune stimuli (
      • Basham T.Y.
      • Nickoloff B.J.
      • Merigan T.C.
      • Morhenn V.B.
      Recombinant gamma interferon induced HLA-DR expression on human keratinocytes.
      ). MHC-II molecules can signal through intracellular calcium, as well as through kinases including protein kinases A and C (PKA and PKC) (
      • Wakita H.
      • Tokura Y.
      • Furukawa F.
      • Takigawa M.
      Staphylococcal enterotoxin B upregulates expression of ICAM-1 molecules on IFN-gamma-treated keratinocytes and keratinocyte cell lines.
      ;
      • Etienne S.
      • Bourdoulous S.
      • Strosberg A.D.
      • Courad P.O.
      MHC class II engagement in brain endothelial cells induces protein kinase A-dependent IL-6 secretion and phosphorylation of cyclic AMP response element-binding protein.
      ). Keratinocyte MHC-II molecules can also trigger the production of cytokines such as TNF-α (
      • Tokura Y.
      • Yagi J.
      • O'Malley M.
      • et al.
      Superantigenic staphylococcal exotoxins induced T cell proliferation in presence of Langerhans cells or class II-bearing keratinocytes and stimulate keratinocytes to produce T-cell-activating cytokines.
      ).

      Bacterial superantigens and the keratinocyte

      The biology of superantigens

      The term “superantigen” was first coined by White et al in 1989 and refers to a group of microbial and viral proteins that differ in several important respects from conventional peptide antigens (
      • White J.
      • Herman A.
      • Pullen A.M.
      • et al.
      The VB-specific superantigen staphylococcal enterotoxin B. stimulation of mature T cells and clonal deletion in neonatal mice.
      ). First, unlike conventional protein antigens, which are taken up and processed by antigen-presenting cells, superantigens exert their effects as globular intact proteins. Similar to peptide antigens, superantigens are presented by class II MHC molecules; however, they do not interact with the MHC peptide β antigen binding groove, but instead bind to conserved amino acid residues that are on the outer walls of the peptide antigen-binding groove. Thus, whereas recognition of conventional peptide antigens by the T cell receptor is restricted by MHC alleles, recognition of superantigens is not generally MHC restricted. Second, superantigens primarily recognize and bind to the variable region of the T cell receptor β chain (VB). This is in contrast to nominal peptide antigens, which require recognition by all five variable elements (i.e., VB, DB, JB, Vα, Jα) of the T cell receptor. Therefore, the responding frequency of a superantigen is several orders of magnitude greater than a conventional peptide antigen.
      The unique ability of a superantigen to bind directly to (and signal through) MHC class II molecules, and cross-link with a large percentage of T cells expressing relevant T cell receptor VB chains, provides an explanation for the potent immune stimulation seen with these molecules (
      • Tiedemann R.E.
      • Fraser J.D.
      Cross-linking of MHC class II molecules by staphylococcal enterotoxin A is essential for antigen-presenting cell and T cell activation.
      ). The prototypical disease due to bacterial superantigens is toxic shock syndrome caused by staphylococcal exotoxins or streptococcal enterotoxins (
      • Deresiewicz R.L.
      Staphylococcal toxic shock syndrome.
      ).

      Superantigens and inflammatory skin diseases

      As outlined above, bacterial superantigens possess several properties that can contribute to disease pathogenesis. Superantigens can bind to and activate MHC class II molecules, whether constitutively expressed on professional antigen presenting cells such as monocytes, Langerhans, or dendritic cells, or on nonprofessional antigen-presenting cells such as the keratinocyte following cytokine stimulation. Significant pathologic effects can be seen due to either local or massive systemic release of protein cytokines as well as other pro-inflammatory mediators. In addition, pro-inflammatory effects would be further amplified by subsequent activation of T cells.
      Of significance for the ability of these proteins to induce cutaneous inflammation, recent evidence suggests that superantigenic stimulation of T cells promotes skin homing by upregulation of the skin homing receptor CLA, a modified selectin that promotes localization of T cells to the skin. Leung et al reported that stimulation of peripheral blood mononuclear cells with bacterial superantigens resulted in a significant increase of CLA+ T cells, but not T cells expressing other homing molecules, in comparison with treatment with other agents including phytohaemogluttinin or anti-CD3 (
      • Leung D.Y.M.
      • Travers J.B.
      • Giorno R.
      • et al.
      Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.
      ). Bacterial toxins were also found to induce IL-12 production in this model system. Of note, induction of toxin-induced CLA expression was blocked by anti-IL-12, and the addition of IL-12 to phytohaemogluttinin-stimulated T cells induced CLA, but not expression of the mucosal homing receptor α e β 7-integrin(
      • Leung D.Y.M.
      • Gately M.
      • Trumble A.
      • et al.
      Bacterial superantigens induce T-cell expression of the skin-homing receptor, the cutaneous lymphocyte-associated antigen, via stimulation of interleukin 12 production.
      ). These data suggest that bacterial toxins induce the expansion of skin-homing CLA+ T cells in an IL-12-dependent manner, and thus may contribute to the development of skin rashes in superantigen-mediated diseases.
      Psoriasis is a chronic inflammatory skin disorder affecting up to 2% of the general population. The characteristic lesion of psoriasis is a well-demarcated erythematous papule or plaque containing hyperproliferating keratinocytes, as well as infiltrating neutrophils, monocytes, and T lymphocytes (
      • Nickoloff B.J.
      The cytokine network in psoriasis.
      ). Though considered an autoimmune disease, considerable evidence suggests an important role for bacteria in the pathogenesis of psoriasis. Colonization and/or infection with Staphylococcus and Streptococcus have been reported to exacerbate psoriasis (
      • Henderson C.A.
      • Highet A.S.
      Acute guttate psoriasis associated with Lancefield Group C and Group G cutaneous streptococcal infections.
      ;
      • Leung D.Y.M.
      • Walsh P.
      • Giorno R.
      • Norris D.A.
      A potential role for superantigens in the pathogenesis of psoriasis.
      ). Superantigenic toxins produced by these bacteria have been implicated in the initiation and/or propagation of psoriasis (reviewed by
      • Leung D.Y.M.
      • Hauk P.
      • Strickland I.
      • Travers J.B.
      • Norris D.A.
      The role of superantigens in human diseases: therapeutic implications for the treatment of skin diseases.
      ). Indeed, a group of psoriatics whose skin disease was worsening were found to be secondarily infected with superantigen-expressing S. aureus, and skin biopsies revealed lesional T cell receptor VB skewing consistent with the pattern of the isolated superantigen (
      • Leung D.Y.M.
      • Walsh P.
      • Giorno R.
      • Norris D.A.
      A potential role for superantigens in the pathogenesis of psoriasis.
      ). Injection of superantigen-stimulated immunocytes into normal skin from subjects with psoriasis xenografted onto an immunodeficient mouse has also been shown to induce clinical, histologic, and immunologic changes consistent with psoriasis (
      • Boehncke W.H.
      • Dressel D.
      • Zollner T.M.
      • Kaufmann R.
      Pulling the trigger on psoriasis.
      ;
      • Wrone-Smith T.
      • Nickoloff B.J.
      Dermal injection of immunocytes induces psoriasis.
      ).
      The most convincing clinical and experimental association between bacterial superantigens and psoriasis, however, is in patients with acute eruptive (guttate) psoriasis (
      • Lewis H.M.
      • Baker B.S.
      • Bokth S.
      • et al.
      Restricted T-cell receptor V beta gene usage in the skin of patients with guttate and chronic plaque psoriasis.
      ;
      • Leung D.Y.M.
      • Travers J.B.
      • Giorno R.
      • et al.
      Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.
      ). Of note, Leung et al demonstrated up to 50% T cell receptor VB2+ T cells in perilesional skin of subjects with acute eruptive psoriasis induced by a Streptococcal pyrogenic exotoxin C (SPEC) expressing Streptococcus (
      • Leung D.Y.M.
      • Travers J.B.
      • Giorno R.
      • et al.
      Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.
      ). Altogether, these studies have implicated bacterial superantigens in both the initiation as well as the worsening of psoriasis.

      The keratinocyte as a target for superantigens

      Due to the lack of MHC II molecules, the resting keratinocyte is not thought to be a direct target for superantigens (
      • Ezepchuk Y.V.
      • Leung D.Y.M.
      • Middleton M.H.
      • et al.
      Staphylococcal toxins and protein A induce cytotoxicity and release of tumor necrosis factor from human keratinocytes.
      ); however, treatment of resting keratinocytes with cytokines such as γ-interferon has been shown to upregulate MHC II molecules (
      • Basham T.Y.
      • Nickoloff B.J.
      • Merigan T.C.
      • Morhenn V.B.
      Recombinant gamma interferon induced HLA-DR expression on human keratinocytes.
      ). Superantigens have been shown to directly bind to and stimulate γ-interferon-treated primary cultures of human and murine keratinocytes as well as epidermal carcinoma cell lines (
      • Nickoloff B.J.
      • Mitra R.S.
      • Green J.
      • et al.
      Accessory cell function of keratinocytes for superantigens. Dependence on lymphocyte function-associated antigen-1/intracellular adhesion molecule-1 interaction.
      ;
      • Strange P.
      • Skov L.
      • Baadsgaard O.
      Interferon-gamma-treated keratinocytes activate T cells in the presence of superantigens: involvement of major histocompatibility complex class II molecules.
      ;
      • Tokura Y.
      • Yagi J.
      • O'Malley M.
      • et al.
      Superantigenic staphylococcal exotoxins induced T cell proliferation in presence of Langerhans cells or class II-bearing keratinocytes and stimulate keratinocytes to produce T-cell-activating cytokines.
      ). Early biochemical events seen as a consequence of MHC II and superantigen interactions in keratinocytes include a transient intracellular calcium mobilization (
      • Wakita H.
      • Tokura Y.
      • Furukawa F.
      • Takigawa M.
      Staphylococcal enterotoxin B upregulates expression of ICAM-1 molecules on IFN-gamma-treated keratinocytes and keratinocyte cell lines.
      ). Superantigens have also been shown to induce the production of TNF-α in keratinocytes in vitro (
      • Tokura Y.
      • Yagi J.
      • O'Malley M.
      • et al.
      Superantigenic staphylococcal exotoxins induced T cell proliferation in presence of Langerhans cells or class II-bearing keratinocytes and stimulate keratinocytes to produce T-cell-activating cytokines.
      ;
      • Ezepchuk Y.V.
      • Leung D.Y.M.
      • Middleton M.H.
      • et al.
      Staphylococcal toxins and protein A induce cytotoxicity and release of tumor necrosis factor from human keratinocytes.
      ). In addition to their well-characterized effects on MHC II molecules, superantigens have also been reported to interact with MHC class I molecules on squamous cell carcinoma cell lines (
      • Haffner A.C.
      • Zepter K.
      • Elmets C.A.
      Major histocompatibility complex class I molecule serves as a ligand for presentation of superantigen staphylococcal enterotoxin B to T cells.
      ). Topical application of superantigens to atopic skin also induces acute eczematous changes and T cell recptor VB skewing (
      • Skov L.
      • Olsen J.V.
      • Giorno R.
      • et al.
      Application of staphylococcal enterotoxin B on normal and atopic skin induces upregulation of T cells via a superantigen-mediated mechanism.
      )

      Hyperreactivity of psoriatic keratinocytes to topical superantigens

      Significant evidence based on finding peripheral blood or skin T cells with TCR VB skewing and the isolation of a superantigen with a relevant TCR VB profile has linked superantigens to either initiatory events in acute guttate psoriasis (
      • Lewis H.M.
      • Baker B.S.
      • Bokth S.
      • et al.
      Restricted T-cell receptor V beta gene usage in the skin of patients with guttate and chronic plaque psoriasis.
      ;
      • Leung D.Y.M.
      • Travers J.B.
      • Giorno R.
      • et al.
      Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.
      ), or to worsening of psoriasis skin disease (
      • Leung D.Y.M.
      • Walsh P.
      • Giorno R.
      • Norris D.A.
      A potential role for superantigens in the pathogenesis of psoriasis.
      ). Recent studies by Travers et al have examined the effects of topical application of nanogram amounts of the staphylococcal superantigens ZEB and TSST-1, and the streptococcal superantigens SPEA and SPEC to tape-abraded skin of psoriatic patients for 48 h (
      • Travers J.B.
      • Hamid Q.A.
      • Norris D.A.
      • et al.
      Epidermal HLA-DR and the enhancement of cutaneous reactivity to superantigenic toxins in psoriasis.
      ). Inflammatory responses to these purified bacterial proteins were seen in both normal subjects as well as in psoriatics; however, patients with psoriasis exhibited significantly increased inflammatory skin responses in comparison with controls, or subjects with atopic dermatitis or lichen planus. Reactivity of psoriatic subjects to topical superantigens correlated with the amount of skin disease, and was greatest in subjects who exhibited the isomorphic (Koebner) phenomenon. Consistent with the clinical hyperactive response, in situ hybridization studies of skin biopsies obtained 6 and 24 h following patch testing with superantigens demonstrated increased epidermal TNF-α mRNA in psoriatics than controls.
      Though significant numbers of mononuclear cells were seen in superantigen-induced skin reactions, surprisingly, skin biopsies from the lesions did not reveal increased numbers of T cells expressing the predicted T cell receptor VB type for the superantigens. This is in contrast to the T cell receptor VB skewing found after application of larger doses of superantigens on the intact skin of subjects with atopic dermatitis (
      • Skov L.
      • Olsen J.V.
      • Giorno R.
      • et al.
      Application of staphylococcal enterotoxin B on normal and atopic skin induces upregulation of T cells via a superantigen-mediated mechanism.
      ). In addition, immunohistochemical studies revealed increased epidermal MHC-II expression in reactions in psoriatics compared with control skin. Given that superantigens have the ability to induce TNF-α production with MHC-II expressing keratinocytes in vitro (
      • Tokura Y.
      • Yagi J.
      • O'Malley M.
      • et al.
      Superantigenic staphylococcal exotoxins induced T cell proliferation in presence of Langerhans cells or class II-bearing keratinocytes and stimulate keratinocytes to produce T-cell-activating cytokines.
      ;
      • Ezepchuk Y.V.
      • Leung D.Y.M.
      • Middleton M.H.
      • et al.
      Staphylococcal toxins and protein A induce cytotoxicity and release of tumor necrosis factor from human keratinocytes.
      ), one explanation for the increased clinical reactivity and increased epidermal TNF-α production found in psoriatic subjects, would be that the superantigens were stimulating cytokine production from MHC-II positive keratinocytes, with T cell accumulation being in response to these keratinocyte-derived cytokines. This would also explain the lack of T cell receptor VB skewing as well. Consistent with this notion that epidermal MHC-II–superantigen interactions were responsible for the enhanced skin reactions in psoriatic subjects, a mutant TSST-1 toxin that does not bind to MHC-II (TSST-1G31S/S32P) did not induce skin inflammation, even at 10-fold higher concentrations than native superantigens (
      • Travers J.B.
      • Hamid Q.A.
      • Norris D.A.
      • et al.
      Epidermal HLA-DR and the enhancement of cutaneous reactivity to superantigenic toxins in psoriasis.
      ). The lack of skin reactions to the mutant TSST-1 also suggest that the exotoxin-induced skin responses were not due to a delayed type hypersensitivity reaction to a conventional antigen.
      The ability of superantigens to stimulate the production of potent pro-inflammatory and cytotoxic cytokines such as TNF-α in MHC-II positive keratinocytes provides an alternative mechanism by which colonization or actual infection with S. aureus can induce skin inflammation. This pathway could be relevant in psoriasis, as psoriatic lesions are often colonized with Staphylococcus and Streptococcus (
      • Leung D.Y.M.
      • Walsh P.
      • Giorno R.
      • Norris D.A.
      A potential role for superantigens in the pathogenesis of psoriasis.
      ;
      • Ezepchuk Y.V.
      • Leung D.Y.M.
      • Middleton M.H.
      • et al.
      Staphylococcal toxins and protein A induce cytotoxicity and release of tumor necrosis factor from human keratinocytes.
      ), and scratching of the skin would be expected to mimic tape-stripping, by both compromising the barrier function as well as activating the epidermis (
      • Nickoloff B.J.
      • Naidu Y.
      Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin.
      ). This alternative mechanism may explain why some psoriatic patients report improvement of their disease activity following treatment with oral antibiotics (
      • Rosenberg E.W.
      • Noah P.W.
      • Zanolli M.D.
      • Skinner Jr., R.B.
      • Bond M.J.
      • Crutcher N.
      Use of rifampin with penicillin and erythromycin in the treatment of psoriasis. Preliminary report.
      ). It may also account for several reports that fail to demonstrate selective expansion of T cell receptor VB in the skin of patients with plaque psoriasis (
      • Schmitt-Egenolf M.
      • Boehncke W.H.
      • Christophers E.
      • Stander M.
      • Sterry W.
      • Type I.
      • Type I.I.
      psoriasis show a similar usage of T-cell receptor variable regions.
      ;
      • Boehncke W.H.
      • Dressel D.
      • Zollner T.M.
      • et al.
      T-cell-receptor repertoire in chronic plaque psoriasis is restricted and lacks enrichment of superantigen-associated V beta regions.
      ). Thus, epicutaneous application of superantigens on psoriatic skin may result in an acute inflammatory, keratinocyte-mediated response rather than superantigen-mediated T cell activation.

      Staphylococcal α-Toxin, Protein A, and the Keratinocyte

      In addition to toxins that can have profound immunomodulatory effects through their abilities to act as superantigens, S. aureus can produce other proteins that can act as virulence factors and affect host responses. The production of transmembrane pores by staphylococcal α-toxin can induce a potent cytotoxic response that also results in cytokine production. Though not associated with a direct cytotoxic response, staphylococcal Protein A can also induce cytokine production in keratinocytes. In contrast to superantigens, both α-toxin and Protein A can interact with resting (i.e., MHC II-negative) keratinocytes.

      The biology of α-toxin

      Staphylococcal α-toxin is released by bacteria as a 293 residue single chain polypeptide. Upon interaction with a target cell, α-toxin forms a heptamer, which results in the formation of a transmembrane pore (
      • Song L.
      • Hobaugh M.R.
      • Shustak C.
      • et al.
      Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore.
      ). Recent studies using biotinylated α-toxin in susceptible versus resistant cells suggest that the signaling and cytotoxic effects are related to the ability of the α-toxin heptamer to form a transmembrane channel (
      • Valeva A.
      • Walev I.
      • Pinkernell M.
      • et al.
      Transmembrane barrel of staphylococcal apha toxin forms in sensitive, but not resistant cells.
      ). Formation of transmembrane pores results in membrane damage as well as an influx of extracellular calcium, a potent damage-related stimulus for many cells. Similar to calcium ionophores such as A23187, small amounts of α-toxin result in signaling and potential repairable damage, whereas larger amounts result in cell death.
      In endothelial cells as well as in PC-12 cells, α-toxin activates PLA2 via a calcium-dependent process (
      • Fink D.
      • Contreras M.L.
      • Leikes P.I.
      • Lazarovici P.
      Staphylococcus aureus alpha-toxin activates phospholipases and induces a Ca2+ influx in PC-12 cells.
      ). In endothelial cells, α-toxin stimulates arachidonic acid release, as well as PAF biosynthesis (
      • Suttorp N.
      • Buerke M.
      • Tannert-Otto S.
      Stimulation of PAF synthesis in pulmonary artery endothelial cells by staphylococcus aureus alpha-toxin.
      ;
      • Grimminger F.
      • Rose F.
      • Sibelius U.
      • et al.
      Human endothelial cell activation and mediator release in response to the bacterial exotoxins escherichia coli hemolysis and staphylococcal alpha-toxin.
      ). Staphylococcal α-toxin has also been reported to enhance IL-6 and G-CSF production in IL-1 β-treated endothelial cell cultures (
      • Soderquist B.
      • Kallman J.
      • Holmberg H.
      • Vikefors T.
      • Kihlstrohm E.
      Secretion of IL-6, IL-8, and G-CSF by human endothelial cells in vitro in response to staphylococcus aureus and staphylococcal exotoxins.
      ). Exposure of isolated perfused rat hearts to α-toxin was shown to induce coronary vasoconstriction and loss of myocardial contractility (
      • Sibelius U.
      • Grandel U.
      • Buerke M.
      • et al.
      Staphylococcal [alpha]-toxin provokes coronary vasoconstriction and loss in mycocardial contractility in perfused rat hearts: role of thromboxane generation.
      ). Significant levels of thromboxane A2 were found in the perfusate following α-toxin treatment. Of note, indomethacin, acetylsalicylic acid, and a specific thromboxane receptor antagonist inhibited α-toxin effects, demonstrating involvement of endogenous thromboxane A2 in this model system. Thus, α-toxin can induce organ damage not only through direct cytolytic effects, but also through the ability to trigger cellular production of pro-inflammatory and cytotoxic soluble mediators.

      The keratinocyte as a target for α-toxin

      Staphylococcus aureus is not only a common agent of cutaneous infections, but can also be isolated from many patients with inflammatory skin diseases such as atopic dermatitis and psoriasis. A recent study by Ezepchuk and colleagues demonstrated that nine of 19 strains of S. aureus obtained from clinical lesions of atopic dermatitis and psoriasis contained α-toxin (
      • Ezepchuk Y.V.
      • Leung D.Y.M.
      • Middleton M.H.
      • et al.
      Staphylococcal toxins and protein A induce cytotoxicity and release of tumor necrosis factor from human keratinocytes.
      ). Ezepchuk et al also found that treatment of primary cultures of human keratinocytes or the keratinocyte-derived cell line HaCaT with α-toxin resulted in significant TNF-α production. Staphyloccoccal α-toxin also induced cytotoxic effects in keratinocytes that were more compatible with necrosis than apoptosis (
      • Walev I.
      • Martin E.
      • Jonas D.
      • et al.
      Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions.
      ;
      • Ezepchuk Y.V.
      • Leung D.Y.M.
      • Middleton M.H.
      • et al.
      Staphylococcal toxins and protein A induce cytotoxicity and release of tumor necrosis factor from human keratinocytes.
      ). Injection of rabbit corneas with α-toxin has been shown to result in significant tissue edema with death of corneal epithelial cells by both necrosis and apoptosis (
      • Moreau J.M.
      • Sloop G.D.
      • Engel L.S.
      • Hill J.M.
      • O'Callaghan R.J.
      Histopathological studies of staphylococcal alpha toxin: effects on rabbit corneas.
      ). Though α-toxin is thought to have considerable deleterious effects, it has been suggested that α-toxin-induced keratinocyte damage can protect against superantigen-mediated effects by inhibiting the ability of the keratinocyte to activate T cells (
      • Tokura Y.
      • Furakawa F.
      • Wakita H.
      • Yagi H.
      • Ushijima T.
      • Takigawa M.
      T-cell proliferation to superantigen-releasing Staphylococcus aureus by MHC Class II-bearing keratinocytes underprotection from bacterial cytolysin.
      ).
      Allapatt et al recently examined the ability of α-toxin to stimulate lipid mediator production in human carcinoma cell lines.
      Allapatt C, Leung DYM, Johnson C, Clay K, Cosgrove J, Travers JB: Acute keratinocyte damage results in the biosynthesis of the lipid mediator platelet-activating factor. J Invest Dermatol 112:543, 1999 (abstr)
      Using select ion monitoring gas chromatography mass spectrometry, significant production of PAF as well as arachidonic acid release was found in α-toxin-treated HaCaT cells. The amount of PAF produced in response to 10 µg per ml α-toxin was about 25% of that seen in response to a potent ionophore such as A23187 (
      • Travers J.B.
      • Huff J.C.
      • Rola-Pleszcynski M.
      • Gelfand E.W.
      • Morelli J.G.
      • Murphy R.C.
      Identification of functional paltelet-activating factor receptors on human keratinocytes.
      ). Because activation of the epidermal PAF-R can induce arachidonic acid release (
      • Pei Y.
      • Barber L.A.
      • Murphy R.C.
      • et al.
      Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.
      ), the potential involvement of endogenous PAF in α-toxin-induced arachidonic acid release was tested using a model system created by retroviral-mediated transduction of the PAF-R-negative epithelial cell line KB with the human PAF-R. As shown in Figure 1, α-toxin stimulated arachidonic acid release in both control (PAF-R-negative) KBM as well as PAF-R-positive KBP cells; however, the amounts of arachidonic acid released by α-toxin-treated KBP cells were significantly greater than with KBM cells (Figure 1). Similarly, pretreatment of KBP cells with 10 µM of the PAF-R antagonist WEB 2086 partially inhibited α-toxin-induced arachidonic acid release in KBP but not KBM cells (data not shown). These data are compatible with the notion that α-toxin can generate both arachidonic acid release and PAF biosynthesis through calcium-mediated activation of PLA2, and that endogenous PAF generated can feed forward to induce further mediator production. Altogether, through its direct effects on keratinocyte membranes, α-toxin can induce the synthesis of both lipid and protein cytokines, as well as cause direct cytotoxic effects.
      Figure thumbnail gr1
      Figure 1Staphylococcal α-toxin stimulates increased arachidonic acid release in PAF-R expressing versus control KB cells. KB cells transduced with the PAF-R (KBP) or control retrovirus (KBM) were stimulated with 10 µg per ml purified α-toxin or vehicle for 30 min, and arachidonic acid released into the supernatant was quantitated by gas chromatography mass spectrometry using deuterated arachidonic acid as internal standard as previously described (
      • Pei Y.
      • Barber L.A.
      • Murphy R.C.
      • et al.
      Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.
      ). The data shown are mean ± standard deviation of arachidonic acid released from duplicate samples from a representative experiment of four conducted.

      The biology of Protein A

      The unique protein derived from the cell wall structure of S. aureus termed Protein A was discovered over 40 y ago. A characteristic property of this protein has been its ability to bind the Fc structure of IgG, making it a valuable laboratory reagent for the isolation and purification of antibody molecules. Over time other biologic properties of this molecule have been elucidated. Protein A appears to have an unrivaled diversity of biologic activities ranging from immunomodulatory and antitumor to antifungal and antiparasitic effects (reviewed by
      • Silverman G.J.B.
      cell superantigens: possible roles in immunodeficiency and autoimmunity.
      ). Protein A has mitogenic effects through its ability to bind to the VH sequence of the B cell receptor, and stimulates T cells by cross-linking MHC II molecules (
      • Berk G.I.
      • Lederman M.M.
      • Liebman M.L.
      • Ellner J.J.
      Staphylococcal protein A primed leukocytes enhance the autologous mixed lymphocyte reaction.
      ;
      • Feijo G.C.
      • Sabbaga J.
      • Carneiro C.R.
      • Brigido M.M.
      Variable region structure and staphylococcal protein A binding specificity of a mouse monoclonal IgM anti-laminin-receptor antibody.
      ). Protein A can also stimulate production of cytokines including γ-interferon and TNF-α in immunocytes as well as hepatocyte growth factor in fibroblasts (
      • Baroni A.
      • Perfetto B.
      • Ruocco E.
      • Rossano F.
      Lipotechoic acid and protein-A from Staphylococcus aureus stimulate release of hepatocyte growth factor by human dermal fibroblasts.
      ;
      • Sinha P.
      • Ghosh A.K.
      • Das TSaG
      • Ray P.K.
      Protein A of staphylococcus aureus evokes a TH1 type response in mice.
      ).

      The keratinocyte as a target for Protein A

      Though Protein A is consistently produced by essentially all strains of S. aureus, little is known about the effects of this protein on keratinocytes. Ezepchuk and colleagues have reported that Protein A is a potent stimulus for TNF-α production in both primary cultures of human keratinocytes and HaCaT cells (
      • Ezepchuk Y.V.
      • Leung D.Y.M.
      • Middleton M.H.
      • et al.
      Staphylococcal toxins and protein A induce cytotoxicity and release of tumor necrosis factor from human keratinocytes.
      ). The mechanism by which Protein A exerts its effects on epithelial cells is not known; however, the finding that it has potent cytokine-producing effects on resting keratinocytes suggests that it is not acting via cross-linking MHC II molecules.
      In contrast to α-toxin, Protein A treatment is not associated with cytotoxic effects in epithelial cells (
      • Ezepchuk Y.V.
      • Leung D.Y.M.
      • Middleton M.H.
      • et al.
      Staphylococcal toxins and protein A induce cytotoxicity and release of tumor necrosis factor from human keratinocytes.
      ). In addition, Protein A does not stimulate a calcium mobilization response, arachidonic acid release, or PAF biosynthesis in resting HaCaT cells (Travers, unpublished data). Given that Protein A can interact with MHC-II molecules in lymphocytes (
      • Berk G.I.
      • Lederman M.M.
      • Liebman M.L.
      • Ellner J.J.
      Staphylococcal protein A primed leukocytes enhance the autologous mixed lymphocyte reaction.
      ;
      • Tiedemann R.E.
      • Fraser J.D.
      Cross-linking of MHC class II molecules by staphylococcal enterotoxin A is essential for antigen-presenting cell and T cell activation.
      ), however, it is possible that this protein could have similar effects on intracellular calcium levels as superantigens exert in MHC II-expressing keratinocytes.

      Therapeutic implications of toxin–keratinocyte interactions

      The skin is a primary site of S. aureus infection and colonization. Indeed, toxins and other bacterial protein products of S. aureus are hypothesized to act as triggers or persistence factors in several inflammatory skin diseases. Thus, an understanding of the interactions of these proteins with target cells has important therapeutic implications. Though more information is available for other cell types, especially lymphocytes, significant evidence is accumulating suggesting that the keratinocyte may be an important target for S. aureus-derived proteins such as superantigens, α-toxin, and Protein A. As shown in Figure 2, keratinocyte interactions with these proteins could result in the production of potent pro-inflammatory mediators such as TNF-α and PAF, as well as direct cytotoxic effects, compromising the barrier function of the skin. The capability of S. aureus proteins to stimulate cutaneous inflammation can explain why the judicial use of antibiotics can have therapeutic potential in the treatment strategies in some patients with inflammatory diseases.
      Figure thumbnail gr2
      Figure 2Interactions between Staphylococcal proteins and the keratinocyte. The keratinocyte has the potential to be a target for S. aureus proteins. Enterotoxins that act as superantigens (SAg) can signal through MHC-II molecules found on activated keratinocytes, resulting in a calcium mobilization response as well as the production of TNF-α. Protein A does not appear to induce a calcium mobilization response, but is a potent inducer of keratinocyte TNF-α production through an unknown mechanism. Through its ability to form a heptameric transmembrane pore, α-toxin can trigger a calcium mobilization response as well as TNF-α production.

      ACKNOWLEDGMENTS

      The authors wish to acknowledge the technical assistance of Dr. Keith Clay, Christopher Johnson, and Yong Pei. These studies were supported by grants the Indiana University Showalter Memorial Research Fund, and the National Institutes of Health grants K081993, HL62996, AR41256, HL37260, and 5M01 RR0051.

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