Cold stress-induced modulation of inflammatory responses and intracerebral cytokine mRNA expression in acute murine toxoplasmosis

The effects of a physical stressor, cold water stress (CWS), within the central nervous system were investigated in the acute phase of infection with Toxoplasma gondii. Female BALB/c mice were subjected to CWS for 5 min each day for 8 days prior to oral infection with 20 cysts of the low virulent ME 49 strain. Animals were killed at 10-day intervals to detect inflammation, gliosis, and expression of intracerebral cytokine mRNAs. Zones of inflammation were detected by Nissl staining and gliosis by immunoreactivity to glial fibrillary acidic protein. Larger zones of inflammation and reactive astrogliosis were consistently observed in mice subjected to CWS and infected (CWS +INF) compared to control infected (INF) mice. Expression of interleukin (IL)-1beta, IL-2, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide synthase (iNOS) were decreased in CWS+INF mice at 10 days postinoculation (PI), followed by a gradual increase after day 20 PI. This was in contrast to increased expression of these cytokines at 10 days PI in INF mice with a gradual decline thereafter. Inflammation and astrogliosis in CWS+INF mice were associated with an increased expression of IL-1beta, IL-6, IL-12, and TNF-alpha between 20 and 30 days PI. These findings correlated with the continuous gene expression of tachyzoite surface antigen (SAG)-1 mRNA in CWS+INF mice compared to its sharp decline in INF mice after 20 days PI. These results suggest that CWS delays regulation and control of intracerebral Toxoplasma gondii during acute infection in BALB/c mice by decreasing the early expression of IFN-gamma, IL-2, TNF-alpha, iNOS, IL-1beta, and IL-12, while increasing the expression of IL-6, a counterregulatory cytokine.     

Amomum xanthiodes inhibits mast cell-mediated allergic reactions through the inhibition of histamine release and inflammatory cytokine production

In this study, we investigated the effect of Amomum xanthiodes (Zingiberaceae) extract (AXE) on the mast cell-mediated allergy model and studied the possible mechanism of action. We found that AXE inhibited compound 48/80-induced systemic reactions and plasma histamine release in mice. Additionally, AXE decreased immunoglobulin E (IgE)-mediated local allergic reactions and passive cutaneous anaphylaxis (PCA), and AXE dose-dependently attenuated the release of histamine from rat peritoneal mast cells (RPMC) activated by compound 48/80 or IgE. The amounts of AXE needed for inhibition of compound 48/80-induced plasma histamine release and PCA were similar to disodium cromoglycate, the known anti-allergic drug. We found that AXE increased the cAMP levels and decreased the compound 48/80-induced intracellular Ca2+. Furthermore, AXE attenuated the phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore (A23187)-stimulated tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 secretion in human mast cells. The inhibitory effect of AXE on the proinflammatory cytokines was nuclear factor-kappaB (NF-kappaB)-dependent. In addition, AXE decreased PMA plus A23187-induced degradation of IkappaBalphaand the nuclear translocation of NF-kappaB. Our findings provide evidence that AXE inhibits mast cell-derived immediate-type allergic reactions, and that cAMP, intracellular Ca2+, proinflammatory cytokines, and NF-kappaB are involved in these effects.     

Cercarial dermatitis caused by bird schistosomes comprises both immediate and late phase cutaneous hypersensitivity reactions

Avian schistosomes are the primary causative agent of cercarial dermatitis in humans, but despite its worldwide occurrence, little is known of the immune mechanism of this disease. Using a murine model, hosts were exposed to primary (1x) and multiple (4x) infections of Trichobilharzia regenti via the pinna. Penetration of larvae into the skin evoked immediate edema, thickening of the exposure site, and an influx of leukocytes, including neutrophils, macrophages, CD4+ lymphocytes, and mast cells. A large proportion of the latter were in the process of degranulating. After 1x infection, inflammation was accompanied by the release of IL-1beta, IL-6, and IL-12p40. In contrast, in 4x reinfected animals the production of histamine, IL-4, and IL-10 was dramatically elevated within 1 h of infection. Analysis of Ag-stimulated lymphocytes from the skin-draining lymph nodes revealed that cells from 1x infected mice produced a mixed Th1/Th2 cytokine response, including abundant IFN-gamma, whereas cells from 4x reinfected mice were Th2 polarized, dominated by IL-4 and IL-5. Serum Abs confirmed this polarization, with elevated levels of IgG1 and IgE after multiple infections. Infection with radiolabeled cercariae revealed that almost 90% of larvae remained in the skin, and the majority died within 8 days after infection, although parasites were cleared more rapidly in 4x reinfected mice. Our results are the first demonstration that cercarial dermatitis, caused by bird schistosomes, is characterized by an early type I hypersensitivity reaction and a late phase of cutaneous inflammation, both associated with a polarized Th2-type acquired immune response.     

Identification of histamine releasing factor(s) in the late phase of cutaneous IgE-mediated reactions

We have shown that fluids collected from antigen-challenged skin blisters during the late phase reaction cause the release of substantial amounts of histamine (means = 42%, n = 14) from human basophils in vitro. Control fluids collected either during the immediate phase or from an unchallenged blister released less than or equal to 10% histamine from both basophils and lung mast cells. Late phase blister fluids induced low levels of histamine release from human lung cells (means = 11%, n = 4) that were slightly but not significantly greater than levels induced by control blister fluids. The characteristics of basophil release were similar to IgE-mediated stimuli in dose dependence, calcium and temperature requirements, and kinetics. The IgE dependence of the late phase blister fluid was demonstrated by desensitization of the basophils to anti-IgE, which obviated the response to anti-IgE and blister fluid but did not affect a non-IgE-mediated stimulus. Removal of the cell surface IgE with lactic acid also abolished the response to both anti-IgE and late phase blister fluid. Incubation of the "stripped" cells with serum containing IgE myeloma restored the response to anti-IgE but failed to affect response to late phase blister fluid. The characteristics of release obtained with this factor closely resemble those of an IgE-dependent histamine releasing factor from cultured macrophages previously described by our group.     

Role of STAT6 in IgE receptor/FcepsilonRI-mediated late phase allergic responses of mast cells.

In this study we show that IgE receptor engagement triggers activation of STAT6 in mast cells. We sought to determine the role of STAT6 activation in IgE receptor-mediated mast cell responses using STAT6 knockout mice. After IgE receptor engagement, bone marrow mast cells from STAT6(-/-) mice exhibited normal histamine and leukotriene C(4) release, but their cytokine release was markedly reduced. In accordance with these in vitro data, IgE/Ag-challenged STAT6(-/-) mice showed normal early phase, but severely impaired late phase, allergic reactions. These findings provide unprecedented evidence that STAT6 plays a pivotal role in mast cell responses to IgE/Ag stimulation.     

Separation of late bronchial responses from airway hyperresponsiveness in allergic sheep

Allergic sheep with antigen-induced early and late responses were used to determine whether airway hyperresponsiveness (AHR) to carbachol is present during the late response and whether blocking the late response with the leukotriene D4 (LTD4) antagonist MK-571 also blocks this AHR. To do this, we first showed that MK-571 blocked the antigen-induced late response, and then, in a separate study, we determined the effect of MK-571 treatment on airway responsiveness 6 h after antigen challenge (at the start of the late response). MK-571 (5 mg, by metered dose inhaler) given 30 min before and 4 h after Ascaris suum challenge had no effect on the acute response to antigen but blocked (P less than 0.05) the late response compared with placebo (n = 7). In the second study (n = 6), the antigen-induced acute increases in mean specific lung resistance (sRL) were also similar in the placebo (249%) and drug trials (247%). By 6 h postchallenge, however, mean sRL in the placebo trial began to increase (54%, P less than 0.05), whereas in the drug trial mean sRL was baseline. Nevertheless, AHR was apparent in both trials as indicated by a mean twofold leftward shift in the dose-response curves to inhaled carbachol (P less than 0.05 vs. prechallenge). Bronchoalveolar lavage at 6 h showed that MK-571 did not prevent the inflammatory cell influx into the lung. These observations suggest that although LTD4 may be a mediator of the late response in sheep, it is not a primary mediator affecting cholinergic AHR during this period.     

Effects of Prunella vulgaris on mast cell-mediated allergic reaction and inflammatory cytokine production

In this study, we investigated the effect of aqueous extract of Prunella vulgaris (Labiatae; PVAE) on the mast cell-mediated allergy model. We found that PVAE (0.001-0.1 g/kg) dose dependently inhibited compound 48/80-induced systemic anaphylaxis and serum histamine release in mice. PVAE decreased the IgE-mediated local allergic reaction, passive cutaneous anaphylaxis. In addition, PVAE attenuated phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated TNF-alpha, IL-6, and IL-8 secretion in human mast cells. The inhibitory effect of PVAE on proinflammatory cytokines was nuclear factor-kappaB (NF-kappaB) dependent. PVAE suppressed PMA and A23187-induced NF-kappaB/DNA binding activity and NF-kappaB-dependent gene reporter assay. Our findings provide evidence that PVAE inhibits mast cell-derived immediate-type allergic reactions and involvement of proinflammatory cytokines and NF-kappaB in these effects.     

Early and late allergic phase related cough response in sensitized guinea pigs with experimental allergic rhinitis

Cough is a common and important symptom of asthma and allergic rhinitis. Previous experimental evidence has shown enhanced cough sensitivity during early phase of experimental allergic rhinitis in guinea pigs. We hypothesized that airway inflammation during the late phase response after repeated nasal antigen challenge may affect the afferent sensory nerve endings in the larynx and tracheobronchial tree and may also modulate cough response. In the present study we evaluated the cough sensitivity during a period of early and late allergic response in sensitized guinea pigs after repeated nasal antigen challenges. Forty-five guinea pigs were sensitized with ovalbumin (OVA). Four weeks later 0.015 ml of 0.5 % OVA was intranasally instilled to develop a model of allergic rhinitis that was evaluated from the occurrence of typical clinical symptoms. Animals were repeatedly intranasally challenged either by OVA (experimental group) or by saline (controls) in 7-day intervals for nine weeks. Cough was elicited by inhalation of citric acid aerosols. Cough was evaluated at 1 or 3 h after the 6th nasal challenge and 17 or 24 h after the 9th nasal challenge. The cough reflex was significantly increased at 1 and 3 h after repeated nasal challenge in contrast to cough responses evoked at 17 and 24 h after repeated nasal challenge. In conclusion, enhanced cough sensitivity only corresponds to an early allergic response after repeated nasal challenges.     

Activation of toll-like receptor 2 in acne triggers inflammatory cytokine responses

One of the factors that contributes to the pathogenesis of acne is Propionibacterium acnes; yet, the molecular mechanism by which P. acnes induces inflammation is not known. Recent studies have demonstrated that microbial agents trigger cytokine responses via Toll-like receptors (TLRs). We investigated whether TLR2 mediates P. acnes-induced cytokine production in acne. Transfection of TLR2 into a nonresponsive cell line was sufficient for NF-kappa B activation in response to P. acnes. In addition, peritoneal macrophages from wild-type, TLR6 knockout, and TLR1 knockout mice, but not TLR2 knockout mice, produced IL-6 in response to P. acnes. P. acnes also induced activation of IL-12 p40 promoter activity via TLR2. Furthermore, P. acnes induced IL-12 and IL-8 protein production by primary human monocytes and this cytokine production was inhibited by anti-TLR2 blocking Ab. Finally, in acne lesions, TLR2 was expressed on the cell surface of macrophages surrounding pilosebaceous follicles. These data suggest that P. acnes triggers inflammatory cytokine responses in acne by activation of TLR2. As such, TLR2 may provide a novel target for treatment of this common skin disease.     

The roles of IL-4, IL-5 and mast cells in the accumulation of eosinophils during allergic cutaneous late phase reaction in mice

Late phase allergic response has been implicated in the pathogenesis of allergic diseases. In the current study, we investigated the role of IL-4, IL-5 and mast cells in the development of cutaneous late phase reaction (LPR) in mice. Antigenic challenge of ears of ovalbumin (OVA)-immunized BALB/c mice caused a biphasic ear swelling peaking at 1 hr (immediate phase reaction; IPR) and 24 hr (LPR). Ear swelling in LPR was significantly suppressed by the treatment with anti-IL-4 monoclonal antibody (mAb) before antigen challenge. Local eosinophil accumulation during LPR, however, was not inhibited by anti-IL-4 mAb. Moreover, anti-IL-5 mAb had no effect on the swelling response though it significantly suppressed the local accumulation of eosinophils. Interestingly, mast cell-deficient mice (WBB6F1-W/Wv) developed LPR without exhibiting IPR, while the magnitude of ear swelling and local eosinophilia was significantly lower than in normal congenic mice (+/+ mice). The present findings show that IL-4 and IL-5 differently regulate the development of LPR, and that IgE-mediated mast cell activation is required for full response.     

Inhibition of allergic late airway responses by inhaled heparin-derived oligosaccharides.

Inhaled heparin has been shown to inhibit allergic bronchoconstriction in sheep that develop only acute responses to antigen (acute responders) but was ineffective in sheep that develop both acute and late airway responses (LAR) (dual responders). Because the antiallergic activity of heparin is molecular-weight dependent, we hypothesized that heparin-derived oligosaccharides (<2, 500) with potential anti-inflammatory activity may attenuate the LAR in the dual-responder sheep. Specific lung resistance was measured in 24 dual-responder sheep before and serially for 8 h after challenge with Ascaris suum antigen for demonstration of early airway response (EAR) and LAR, without and after treatment with inhaled medium-, low-, and ultralow-molecular-weight (ULMW) heparins and "non-anticoagulant" fractions (NAF) of heparin. Airway responsiveness was estimated before and 24 h postantigen as the cumulative provocating dose of carbachol that increased specific lung resistance by 400%. Only ULMW heparins caused a dose-dependent inhibition of antigen-induced EAR and LAR and postantigen airway hyperresponsiveness (AHR), whereas low- and medium-molecular-weight heparins were ineffective. The effects of ULMW heparin and ULMW NAF-heparin were comparable and inhibited the LAR and AHR even when administered "after" the antigen challenge. The ULMW NAF-heparin failed to inhibit the bronchoconstrictor response to histamine, carbachol, and leukotriene D(4), excluding a direct effect on airway smooth muscle. In six sheep, segmental antigen challenge caused a marked increase in bronchoalveolar lavage histamine, which was not prevented by inhaled ULMW NAF-heparin. The results of this study in the dual-responder sheep demonstrate that 1) the antiallergic activity of inhaled "fractionated" heparins is molecular-weight dependent, 2) only ULMW heparins inhibit the antigen-induced EAR and LAR and postantigen AHR, and 3) the antiallergic activity is mediated by nonanticoagulant fractions and resides in the ULMW chains of <2,500.     

The biologic activity of mast cell granules. IV. The effect of complement depletion on rat cutaneous late phase reactions

Cutaneous late phase reactions (LPR) in rats can be induced by the intradermal injection of anti-IgE antibody or isolated rat peritoneal mast cell granules. Rat LPR are characterized by neutrophil-rich infiltrates at 2 to 8 hr followed by mononuclear cell-rich infiltrates thereafter. Rat Arthus reactions are histologically similar and are complement (C) dependent. To determine the importance of C in the pathogenesis of rat LPR compared with its role in Arthus reactions, rats were treated with cobra venom factor (CVF) (250 U/kg i.v.), and the effects of this treatment on total hemolytic complement (CH50), C3 titers, LPR, and Arthus reactions were assessed. CVF treatment produced profound decreases in both CH50 (from 197 +/- 20 to less than 1.0 U/ml) and C3 (from 44,240 +/- 2840 to less than 5 U/ml) titers after 6 hr, which persisted through at least 30 hr. The inflammatory intensity of heterologous reverse passive Arthus reactions was significantly decreased in CVF-treated animals. In contrast, the intensity of LPR was unaffected by CVF treatment. Therefore, although LPR and Arthus reactions share certain histologic characteristics, these similarities are not due to a mutual requirement for the presence of C.     

Production of early and late pulmonary responses with inhaled leukotriene D4 in allergic sheep

Some allergic sheep respond to inhalation of Ascaris suum antigen with both immediate and late increases in airflow resistance (late response). The mechanism of the late response is unknown but recent evidence suggests that the initial generation of slow-reacting substance of anaphylaxis (SRS-A) immediately after antigen challenge is a necessary pre-requisite for the physiologic expression of this late response. Based on this evidence we hypothesized that airway challenge with leukotriene D4 (LTD4), an active component of SRS-A would produce acute and late airway responses in allergic sheep similar to those observed with antigen. In five allergic sheep with documented early and late pulmonary responses to Ascaris suum antigen, inhalation of leukotriene D4 aerosol (delivered dose (mean +/- SE) 0.55 +/- 0.08 ug) resulted in significant early and late increases in specific lung resistance (SRL). In three allergic sheep which only demonstrated acute responses to antigen, LTD4 aerosol (delivered dose 0.59 +/- 0.09 ug) only produced an acute increase in SRL. In the late responders pretreatment with aerosol cromolyn sodium (1 mg/kg) did not affect the acute response but blunted the late increase in SRL. Pretreatment with aerosol FPL-57231 (1% w/v solution) completely blocked both the acute and late responses. These data support the hypothesis that initial release of LTD4 in the airways of sensitive animals is important for the physiologic expression of the late response.     

Noncanonical notch signaling modulates cytokine responses of dendritic cells to inflammatory stimuli

Dendritic cell (DC)-derived cytokines play a key role in specifying adaptive immune responses tailored to the type of pathogen encountered and the local tissue environment. However, little is known about how DCs perceive the local environment. We investigated whether endogenous Notch signaling could affect DC responses to pathogenic stimuli. We demonstrate that concurrent Notch and TLR stimulation results in a unique cytokine profile in mouse bone-marrow derived DCs characterized by enhanced IL-10 and IL-2, and reduced IL-12 expression compared with TLR ligation alone. Unexpectedly, modulation of cytokine production occurred through a noncanonical Notch signaling pathway, independent of γ-secretase activity. Modulation required de novo protein synthesis, and PI3K, JNK, and ERK activity were necessary for enhanced IL-2 expression, whereas modulation of IL-10 required only PI3K activity. Further, we show that this γ-secretase-independent Notch pathway can induce PI3K activity. In contrast, expression of the canonical Notch target gene Hes1 was suppressed in DCs stimulated with Notch and TLR ligands simultaneously. Thus, our data suggest that Notch acts as an endogenous signal that modulates cytokine expression of DCs through a noncanonical pathway and therefore has the potential to tailor the subsequent adaptive immune response in a tissue- and/or stage-dependent manner.     

Suppressor of cytokine signaling 1 DNA administration inhibits inflammatory and pathogenic responses in autoimmune myocarditis

Myocarditis and subsequent dilated cardiomyopathy are major causes of heart failure in young adults. Myocarditis in humans is highly heterogeneous in etiology. Recent studies have indicated that a subgroup of myocarditis patients may benefit from immune-targeted therapies, because autoimmunity plays an important role in myocarditis as well as contributing to the progression to cardiomyopathy and heart failure. Suppressor of cytokine signaling (SOCS) 1 plays a key role in the negative regulation of both TLR- and cytokine receptor-mediated signaling, which is involved in innate immunity and subsequent adaptive immunity. In this study, we investigated the therapeutic effect of SOCS1 DNA administration on experimental autoimmune myocarditis (EAM) in mice. EAM was induced by s.c. immunization with cardiac-specific peptides derived from α myosin H chain in BALB/c mice. In contrast to control myocarditis mice, SOCS1 DNA-injected mice were protected from development of EAM and heart failure. SOCS1 DNA administration was effective for reducing the activation of autoreactive CD4(+) T cells by inhibition of the function of Ag-presenting dendritic cells. Our findings suggest that SOCS1 DNA administration has considerable therapeutic potential in individuals with autoimmune myocarditis and dilated cardiomyopathy.     

Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2

Human cytomegalovirus (CMV) is a ubiquitous opportunistic pathogen that causes significant morbidity and mortality in immunocompromised people. An understanding of how CMV induces and circumvents host immunity is of critical importance in efforts to design effective therapeutics. It was recently discovered that mere cell contact by CMV particles leads to profound modulation of cellular gene expression, including induction of inflammatory cytokines and interferon-stimulated genes characteristic of innate immune detection. These findings suggest that a membrane receptor recognizes a CMV envelope protein(s), leading to innate immune activation. Here, we show that the pattern recognition receptors Toll-like receptor 2 (TLR2) and CD14 recognize CMV virions and trigger inflammatory cytokine production. Induction of inflammatory cytokines is mediated via TLR2-dependent activation of NF-kappa B. Since many of the pathological processes associated with CMV disease are facilitated or directly mediated by inflammatory cytokines, identification of the host membrane detection machinery may ultimately lead to improved therapeutics.     

Impaired IFN-gamma-dependent inflammatory responses in human keratinocytes overexpressing the suppressor of cytokine signaling 1

Receptors for the bacterial chemotactic peptide fMLP are implicated in inflammation and host defense against microbial infection. We investigated the expression and function of fMLPR in microglial cells, which share characteristics of mononuclear phagocytes and play an important role in proinflammatory responses in the CNS. The expression of the genes encoding formyl peptide receptor (FPR)1 and FPR2, the high- and low-affinity fMLPR, was detected in a murine microglial cell line N9, but these cells did not respond to chemotactic agonists known for these receptors. N9 cells incubated with bacterial LPS increased the expression of fMLPR genes and developed a species of specific, but low-affinity, binding sites for fMLP, in association with marked calcium mobilization and chemotaxis responses to fMLP in a concentration range that typically activated the low-affinity receptor FPR2. In addition, LPS-treated N9 cells were chemoattracted by two FPR2-specific agonists, the HIV-1 envelope-derived V3 peptide, and the 42 aa form of the amyloid beta peptide which is a pathogenic agent in Alzheimer's disease. Primary murine microglial cells also expressed FPR1 and FPR2 genes, but similar to N9 cells, exhibited FPR2-mediated activation only after LPS treatment. In contrast to its effect on the function of FPR2, LPS reduced N9 cell binding and biological responses to the chemokine stromal cell-derived factor-1alpha. Thus, LPS selectively modulates the function of chemoattractant receptors in microglia and may promote host response in inflammatory diseases in the CNS.