Mechanisms of chemical-induced innate immunity in allergic contact dermatitis.

Allergic contact dermatitis (ACD) is one of the most prevalent occupational skin diseases and causes severe and long-lasting health problems in the case of chronification. It is initiated by an innate inflammatory immune response to skin contact with low molecular weight chemicals that results in the priming of chemical-specific, skin-homing CD8(+) Tc1/Tc17 and CD4(+) Th1/Th17 cells. Following this sensitization step, T lymphocytes infiltrate the inflamed skin upon challenge with the same chemical. The T cells then exert cytotoxic function and secrete inflammatory mediators to produce an eczematous skin reaction. The recent characterization of the mechanisms underlying the innate inflammatory response has revealed that contact allergens activate innate effector mechanisms and signalling pathways that are also involved in anti-infectious immunity. This emerging analogy implies infection as a potential trigger or amplifier of the sensitization to contact allergens. Moreover, new mechanistic insights into the induction of ACD identify potential targets for preventive and therapeutic intervention. We summarize here the latest findings in this area of research.

Innate and adaptive immune responses in contact dermatitis: analogy with infections.

Allergic contact dermatitis (ACD) is an inflammatory skin disease of great and steadily increasing importance as an occupational health problem. The disease is induced by chemicals and metal ions which penetrate the skin and form complexes with host proteins. This process is accompanied by a strong, allergen-induced inflammatory reaction and leads to the migration of allergen-carrying dendritic cells (DC) from the skin to regional lymph nodes, where they promote generation of allergen-specific T cells. The latter are the ultimate effector cells of the disease. Re-exposure to the causative agent leads to the recruitment of the T effector cells, which then elicit the typical skin inflammatory reaction at the site of contact. Although DC and effector T cells play a protagonistic role in the sensitization and elicitation phase of ACD, respectively, other cell types including keratinocytes, NK cells, mast cells and B cells contribute to the pathogenesis of the disease. In this review the authors summarize recent findings that identify stress responses and innate immune pathways triggered by contact allergens and review recent data regarding the adaptive T cell response. The new data were collected mainly from studies on contact hypersensitivity (CHS), the corresponding experimental mouse model of human ACD. The elucidation of the molecular events involved in contact allergen-induced innate responses will help to design new treatment strategies and may allow to develop predictive in vitro assays for the identification of contact allergens.

Immunotherapy of patients with hormone-refractory prostate carcinoma pre-treated with interferon-gamma and vaccinated with autologous PSA-peptide loaded dendritic cells--a pilot study.

PURPOSE: We conducted a pilot trial to assess the feasibility and tolerability of a prime/boost vaccine strategy using interferon-gamma (IFN-gamma) and autologous dendritic cells (DCs) pulsed with HLA-A2-specific prostate-specific antigen (PSA) peptides (PSA-1 [141-150]; PSA-2 [146-156]; PSA-3 [154-163]) for the treatment of 12 patients with hormone refractory prostate carcinoma. PATIENTS AND METHODS: All patients were vaccinated four times with intracutaneously injected PSA-peptide loaded DCs after subcutaneous administration of IFN-gamma 2 hr before DC administration (50 microg/m(2) body surface). Objectives were safety, clinical benefit, clinical and biochemical response, quality of life, and immunological parameters. RESULTS: The vaccination was well tolerated without any vaccination-associated adverse events. One partial and one mixed responder were identified, four patients showed stable diseases. Two patients had a decrease and four a slow-down velocity slope in the PSA serum level. All responders showed a positive DTH-response, but only two a slight increase in PSA-peptide specific T-lymphocytes. CONCLUSION: The immunotherapy with IFN-gamma and PSA-peptide loaded DCs was feasible and well tolerated. The observed responses imply a potential antitumor activity.

Differential clearance and induction of host responses by various administered or released lipopolysaccharides.

The clearance and activity of different types of lipopolysaccharide (LPS) released during infection with Gram-negative bacteria were investigated. When highly purified preparations differing in their specific endotoxin activity were administered intravenously to mice, the clearance of rough (R)-form LPS preparations from Salmonella minnesota and Escherichia coli was much faster than that of a smooth (S)-form LPS preparation from Salmonella abortus equi, but slower than that of lipo-oligosaccharides (LOS) preparations from Bordetella pertussis and Helicobacter pylori. After intraperitoneal infection with 10(7) and 10(8) CFU E. coli O111:B4, relatively high levels of LPS were detected dose-dependently in the plasma of infected mice and persisted for a long time. In addition, plasma sCD14 levels in infected mice were higher than in LPS-administered mice. These results indicate that continuously higher levels of plasma LPS followed by stronger host responses occur during infection and suggest that these differences between LPS-administered and infected mice should be taken into consideration when analyzing host responses induced by LPS.

Differences in innate defense mechanisms in endotoxemia and polymicrobial septic peritonitis.

Loss, reduction, or enhancement of the ability to respond to bacterial lipopolysaccharide (LPS) has no influence on survival of mice in a model of postoperative polymicrobial septic peritonitis induced by cecal ligation and puncture (CLP). This was demonstrated by using either mice with a defective Tlr4 gene, which encodes the critical receptor molecule for LPS responses, or mice deficient for LPS binding protein (LBP) or mice sensitized to LPS by Propionibacterium acnes. Though interleukin-12 (IL-12) and gamma interferon (IFN-gamma) play an important role in the sensitivity to LPS as well as in the resistance to several infections, loss of these cytokine pathways does not affect survival after CLP. Thus, neutralization of neither endogenous IL-12 nor IFN-gamma altered mortality. In addition, IFN-gamma receptor-deficient mice demonstrated the same sensitivity to CLP as mice with a functional IFN-gamma receptor. However, administration of IFN-gamma at the time of operation or pretreatment of both IFN-gamma-sensitive and IFN-gamma-resistant mice with IL-12 significantly enhanced mortality. This indicates that in the present infection model activation of innate defense mechanisms is not dependent on LPS recognition and does not require endogenous IL-12 or IFN-gamma function. Indeed, exogenous application of these two mediators had deleterious effects.

A point mutation in the IL-12R beta 2 gene underlies the IL-12 unresponsiveness of Lps-defective C57BL/10ScCr mice.

Lps-defective C57BL/10ScCr (Cr) mice are homozygous for a deletion encompassing Toll-like receptor 4 that makes them refractory to the biological activity of LPS. In addition, these mice exhibit an inherited IL-12 unresponsiveness resulting in impaired IFN-gamma responses to different microorganisms. By positional cloning methods, we show here that this second defect of Cr mice is due to a mutation in a single gene located on mouse chromosome 6, in close proximity to the Igkappa locus. The gene is IL-12Rbeta2. Cr mice carry a point mutation creating a stop codon that is predicted to cause premature termination of the translated IL-12Rbeta2 after a lysine residue at position 777. The truncated beta2 chain can still form a heterodimeric IL-12R that allows phosphorylation of Janus kinase 2, but, unlike the wild-type IL-12R, can no longer mediate phosphorylation of STAT4. Because the phosphorylation of STAT4 is a prerequisite for the IL-12-mediated induction of IFN-gamma, its absence in Cr mice is responsible for their defective IFN-gamma response to microorganisms.

TNF-alpha hyper-responses to Gram-negative and Gram-positive bacteria in Propionibacterium acnes primed or Salmonella typhimurium infected mice.

IFN-gamma-dependent hypersensitivity to LPS is inducible in mice by infection or pre-treatment with killed bacteria. Hypersensitive mice exhibit enhanced inflammatory responses to LPS, including the overproduction of TNF-alpha. Using Lps(n) BALB/c and Lps(d) BALB/c/l mice, primed with Propionibacterium acnes or infected with Salmonella typhimurium, we show that concurrently to hypersensitivity to LPS, a hypersensitivity to other constituents of killed Gram-negative or Gram-positive bacteria and to staphylococcal enterotoxin B (SEB) develops. The TNF-alpha hyper-responses in sensitized mice induced by different Gram-positive bacteria, are generally weaker than those by Gram-negative bacteria and vary significantly, due to the absence of a common, LPS-equivalent component. Using IFN-gamma R(-/-) and the respective wild-type mice, we demonstrate that although sensitization to LPS and killed Listeria monocytogenes is exclusively IFN-gamma-dependent, an IFN-gamma-independent, moderate sensitization to certain TNF-alpha-inducing constituents in bacteria may develop in parallel.

Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.

Lipopolysaccharide is an important recognition marker by virtue of which the innate immune system senses and reacts against Gram-negative bacteria invading the LPS susceptible host. This review deals with the factors affecting LPS susceptibility and with the role of the latter in the course and outcome of Salmonella typhimurium infection.

Inherited IL-12 unresponsiveness contributes to the high LPS resistance of the Lps(d) C57BL/10ScCr mouse.

LPS(d) mouse strains are characterized by the presence of a defective LPS/tlr4 gene that make them refractory to the biological activity of LPS. One of the mouse strains commonly used to study LPS defects is the C57BL/10ScCr (Cr) strain. However, unlike other LPS(d) strains, the Cr strain also has a heavily impaired IFN-gamma response to micro-organisms. As a consequence, unlike other LPS(d) mouse strains, they do not acquire a partial LPS susceptibility when treated with sensitizing bacteria. Because IL-12 is important for the microbial induction of IFN-gamma, we investigated whether the production or function of IL-12 might be defective in Cr mice. IL-12 mRNA (p35 and p40) was present in the spleen of untreated Cr mice, IL-12p40 mRNA was inducible in mice injected with live or killed Salmonella typhimurium, and IL-12 (p70) was inducible in macrophages by bacteria. Thus, Cr mice exhibit normal IL-12 responses. In functional tests, splenocytes of untreated or of S. typhimurium-infected mice failed to produce IFN-gamma when stimulated with murine rIL-12 or with a combination of IL-12 and murine rIL-18 or Con A. Furthermore, Cr mice were identical with IL-12p35/p40 and IL-12 receptor beta(1) knockout mice in their impaired in vivo and in vitro IFN-gamma responses to bacteria. Thus, Cr mice carry a second genetic defect unrelated to the Lps/tlr4 mutation that underlies the IL-12 unresponsiveness and contributes to the LPS resistance and impaired innate immune response in this strain.

Bacterial induction of beta interferon in mice is a function of the lipopolysaccharide component.

We investigated the reason for the inability of lipopolysaccharide (LPS)-resistant (Lps-defective [Lps(d)]) C57BL/10ScCr mice to produce beta interferon (IFN-beta) when stimulated with bacteria. For this purpose, the IFN-beta and other macrophage cytokine responses induced by LPS and several killed gram-negative and gram-positive bacteria in LPS-sensitive (Lps-normal [Lps(n)]; C57BL/10ScSn and BALB/c) and Lps(d) (C57BL/10ScCr and BALB/c/l) mice in vitro and in vivo were investigated on the mRNA and protein levels. In addition, double-stranded RNA (dsRNA) was used as a nonbacterial stimulus. LPS and all gram-negative bacteria employed induced IFN-beta in the Lps(n) mice but not in the Lps(d) mice. All gram-positive bacteria tested failed to induce significant amounts of IFN-beta in all four of the mouse strains used. As expected, all other cytokines tested (tumor necrosis factor alpha, interleukin 1alpha [IL-1alpha], IL-6, and IL-10) were differentially induced by gram-negative and gram-positive bacteria. Stimulation with dsRNA induced IFN-beta and all other cytokines mentioned above in all mouse strains, regardless of their LPS sensitivities. The results suggest strongly that LPS is the only bacterial component capable of inducing IFN-beta in significant amounts that are readily detectable under the conditions used in this study. Consequently, in mice, IFN-beta is inducible only by gram-negative bacteria, but not in C57BL/10ScCr or other LPS-resistant mice.

Culture and biological activity of Propionibacterium acnes.

Administration of killed Propionibacterium acnes to experimental animals leads to the development of hypersensitivity to the lethal and cytokine-inducing effects of endotoxin. This sensitizing property of P. acnes is not always expressed by different bacterial preparations. Its expression depends very much on the conditions employed for the cultivation of this microorganism. The present study investigates which culturing conditions result in P. acnes preparations with optimal sensitizing properties. The composition of the medium, the culturing time and temperature as well as the type of cultivation (in minifermentor or stationary culture) were all varied for this purpose. The resulting bacterial preparations were killed at 65 degrees C for 1 h and tested for sensitizing activity. The results show that stationary cultures of P. acnes grown at 37 degrees C for 4 to 5 days in the appropriate medium produce biologically active preparations with satisfactory sensitizing activity.

Induction of CD14 expression in Lpsn, Lpsd and tumor necrosis factor receptor-deficient mice.

The involvement of CD14 in lipopolysaccharide (LPS) recognition and signaling has been demonstrated in several studies. For this reason, we investigated whether the resistance of Lpsd mice to LPS might be related to an impaired CD14 expression. We compared the in vivo and in vitro expression of CD14 in Lpsn (LPS sensitive) and Lpsd mice, and its modulation by LPS, killed gram-negative and gram-positive bacteria and double-stranded (ds)RNA. Untreated Lpsn and Lpsd cultured macrophages (M phi), expressed similar amounts of CD14 mRNA and membrane-bound (m)CD14. LPS enhanced CD14 expression only in Lpsn M phi, while all bacteria, or dsRNA, enhanced CD14 in Lpsn and Lpsd M phi. Similarly, in vivo administration of LPS induced or enhanced CD14 mRNA in different organs of Lpsn mice only, while bacteria or dsRNA in both types of mouse. Furthermore, exogenous recombinant tumor necrosis factor (TNF) induced in vivo and in vitro enhanced CD14 expression in Lpsn, Lpsd and also in TNF receptor 2-deficient (TNFR2-/-) mice, but failed to do so in TNFR1-/- mice, showing that TNFR1 mediates the effect of TNF on CD14. However, LPS, bacteria and dsRNA induced CD14 in both TNFR2-/- and TNFR1-/- mice to a similar extent, revealing that this induction does not require TNF signaling.

Tumor necrosis factor-induced hepatic DNA fragmentation as an early marker of T cell-dependent liver injury in mice.

BACKGROUND & AIMS: The injection of mice with anti-CD3 monoclonal antibody causes activation of T lymphocytes and leads to a lethal shock syndrome. The aim of this study was to investigate T cell-dependent, cytokine-mediated target cell death that leads to organ injury. METHODS: Anti-CD3 monoclonal antibody or staphylococcal enterotoxin B was injected into mice sensitized by D-galactosamine. Liver injury was assessed biochemically and histologically, and circulating cytokines were determined. RESULTS: Mice sensitized with D-galactosamine developed severe liver injury within 8 hours after injection of anti-CD3 monoclonal antibody or staphylococcal enterotoxin B. Apoptotic bodies and chromatin condensation were detectable 5 hours after anti-CD3 monoclonal antibody challenge. DNA fragmentation in the liver preceded the increase in plasma alanine aminotransferase activity. Anti-CD3 monoclonal antibody induced the release of tumor necrosis factor and other cytokines. Passive immunization against tumor necrosis factor or pretreatment with immunosuppressive drugs protected mice from liver injury. Liver injury associated with apoptotic cell death and DNA fragmentation was also noted in D-galactosamine-sensitized mice injected with staphylococcal enterotoxin B. CONCLUSIONS: Tumor necrosis factor-induced hepatic apoptosis followed by necrosis may represent a general pathomechanism of T-cell shock models using D-galactosamine-sensitized mice.

Interferon beta, a cofactor in the interferon gamma production induced by gram-negative bacteria in mice.

The interferon (IFN) gamma production of splenocytes from closely related C57BL/10ScSn (Sn) and C57BL/10ScCr (Cr) mice was compared. Concanavalin A and CD3 monoclonal antibodies induced high levels of IFN-gamma in both Sn and Cr splenocytes. By contrast, treatment with gram-negative bacteria induced IFN-gamma only in Sn splenocytes; in Cr splenocytes, the IFN-gamma response was heavily impaired. The IFN-gamma induction by bacteria requires the cooperation of IFN-gamma-producing cells with macrophages. Depletion of macrophages from Sn splenocytes resulted in the loss of ability to produce IFN-gamma after bacterial stimulation. Reconstitution with new Sn macrophages restored the IFN-gamma responsiveness, whereas reconstitution with Cr macrophages failed to do so. Normal function of IFN-gamma-producing cells and a defective function of macrophages of Cr mice was demonstrated by evidence showing that whole or macrophage-depleted Cr splenocytes, when supplemented with Sn macrophages, acquire the ability to produce IFN-gamma in response to bacteria. A similar effect was achieved by supplementing Cr splenocytes with supernatants of bacteria-stimulated Sn macrophages or with recombinant murine IFN-beta or IFN-alpha. Preincubation of active macrophage supernatants with antibodies to IFN-beta suppressed the helper activity for Cr splenocytes. Moreover, the bacteria-induced production of IFN-gamma by Sn splenocytes could be inhibited by antibodies to murine IFN-beta. The results provide evidence that IFN-beta is an important cofactor of IFN-gamma induction, which is not induced in Cr mice by gram-negative bacteria.

Discordant adaptation of human peritoneal macrophages to stimulation by lipopolysaccharide and the synthetic lipid A analogue SDZ MRL 953. Down-regulation of TNF-alpha and IL-6 is paralleled by an up-regulation of IL-1 beta and granulocyte colony-stimulating factor expression.

Human peritoneal macrophages were exposed to increasing doses of LPS or a synthetic lipid A analogue (SDZ MRL 953) and production of the cytokines IL-1 beta, IL-6, TNF-alpha, and G-CSF was assessed at the protein and mRNA level. Cells were also prestimulated with low doses of LPS and SDZ MRL 953 to study their adaptation to a secondary challenge with high doses of LPS. The ability of macrophages to produce high levels of TNF-alpha and IL-6 after stimulation with LPS could be relieved almost completely by preincubating cells with low doses of LPS. Decreases of TNF-alpha and IL-6 production resulted from inhibition of gene transcription and/or changes in mRNA stability, as transcript levels of these cytokines were down-modulated by the process of LPS adaptation. Surprisingly, however, adapted cells were able to synthesize even larger quantities of G-CSF and IL-1 beta when exposed to a secondary LPS challenge. mRNA levels of the adapted cells remained unaltered for IL-1 beta, but were slightly increased for G-CSF as assessed by Northern blot analysis. High doses of the synthetic lipid A analogue SDZ MRL 953 were also able to adapt macrophages to a secondary LPS challenge by down-regulating TNF-alpha and IL-6 production, whereas priming secretion of G-CSF and IL-1 beta as well. We describe here the discordant adaptation of human peritoneal macrophages to a secondary LPS stimulus in vitro. These findings appear to have ramifications for the in vivo endotoxin response during inflammation and also Gram-negative septicemia.

Contribution of tumor necrosis factor-alpha and glucocorticoid in hydrazine sulfate-mediated protection against endotoxin lethality.

Hydrazine sulfate pretreatment has previously been shown in our laboratory to protect normal mice against endotoxin and D-galactosamine-sensitized mice against both exogenous tumor necrosis factor (TNF) and endotoxin. An intact pituitary is required for manifestation of the protective effects. Further, we have demonstrated that hydrazine sulfate pretreatment specifically modulates the TNF response to lipopolysaccharide (LPS) in mouse macrophages in vitro. This in vivo study was performed to test whether a reduced TNF response and/or increased glucocorticoid response may contribute to hydrazine sulfate protection against LPS-induced lethality in vivo. The results presented here establish that hydrazine sulfate pretreatment selectively attenuates circulating TNF levels following LPS challenge. Moreover, adrenalectomy abrogates hydrazine sulfate protection but does not prevent hydrazine sulfate attenuation of circulating TNF levels in response to LPS. Hydrazine sulfate-mediated protection is, however, restored permissively by corticosterone. Thus, the mechanism by which hydrazine sulfate protects against LPS lethality in adrenalectomized mice includes TNF modulation in response to endotoxin, as well as a pivotal requirement for glucocorticoid.

[The role of cytokines in endotoxic shock and in endotoxin hypersensitivity]. / Die Bedeutung von Zytokinen beim Endotoxinschock und bei der Endotoxinüberempfindlichkeit.

Endotoxins (lipopolysaccharides, LPS) are constituents of the outer membrane of gram-negative bacteria. The application of purified LPS to experimental animals leads to the development of many pathophysiological activities which are also seen during infection with gram-negative microorganisms. One important prerequisite for the development of endotoxin shock is the interaction of LPS with macrophages, the activation of which leads to the release of cytokines, in particular of TNF alpha and IL-1, which mediate the toxic activity of LPS. The interaction of LPS with target cells and the subsequent initiation of endotoxin shock may be modified by plasma proteins that are capable of binding LPS. The most important LPS-binding proteins known so far are high density lipoprotein (HDL), low density lipoprotein (LDL), LPS-binding protein (LBP) and specific LPS antibodies. Native LPS released from bacteria may be associated partly with bacterial proteins (e.g. OmpA) which may also modify its interaction with host targets. The sensitivity of the organism to LPS is genetically determined, but may be altered under different experimental conditions. Hypersensitivity to LPS may be achieved by treatment with live (infection) or killed microorganisms, growing tumors, hepatotoxic agents or treatment with proteins to which the organism has been immunologically primed. The state of hypersensitivity is characterized by an increased ability of hypersensitive animals to produce cytokines on LPS challenge, as well as by an increased susceptibility to the toxic activity of TNF alpha. Recently it could be shown that interferon gamma (IFN gamma) is a central mediator in the development of the hypersensitivity to LPS induced by infection.

Mechanisms of endotoxin shock and endotoxin hypersensitivity.

Endotoxins (lipopolysaccharide, LPS) are biologically active substances present in Gram-negative bacteria. Injection of purified LPS into experimental animals leads to the development of many biological activities that can lead to shock with lethal outcome. The biological activities of LPS are not direct effects of the LPS molecule since LPS usually expresses no direct cytotoxic activity. The toxic and other biological properties of LPS are caused indirectly through the action of endogenous mediators that are formed following interaction of LPS with cellular targets, macrophages occupying a key position in the development of endotoxin shock. The interaction of LPS with macrophages may proceed directly leading to activation of these cells, with subsequent synthesis and secretion of a number of endogenous mediators which initiate the different biological activities of LPS. Tumor necrosis factor alpha (TNF-alpha), a macrophage derived cytokine, is a primary mediator of the lethal action of endotoxin. Sensitivity to LPS is genetically determined, varying considerably among different species. The sensitivity of normal animals (mice) to endotoxin may be enhanced considerably under different experimental conditions that include treatment with live (infection) or killed Gram-negative and -positive bacteria. Sensitization to endotoxin proceeds in all LPS-responder strains investigated and in the LPS-resistant mice of the strain C3H/HeJ. It does not proceed in a second LPS-resistant strain, C57BL/10ScCr. The absence of sensitization in the latter mice was found to be due to an impaired IFN-gamma production. IFN-gamma could be identified as the mediator of endotoxin hypersensitivity induced by bacteria.