Lactic acid suppresses IgE-mediated mast cell function in vitro and in vivo.

Mast cells have functional plasticity affected by their tissue microenvironment, which greatly impacts their inflammatory responses. Because lactic acid (LA) is abundant in inflamed tissues and tumors, we investigated how it affects mast cell function. Using IgE-mediated activation as a model system, we found that LA suppressed inflammatory cytokine production and degranulation in mouse peritoneal mast cells, data that were confirmed with human skin mast cells. In mouse peritoneal mast cells, LA-mediated cytokine suppression was dependent on pH- and monocarboxylic transporter-1 expression. Additionally, LA reduced IgE-induced Syk, Btk, and ERK phosphorylation, key signals eliciting inflammation. In vivo, LA injection reduced IgE-mediated hypothermia in mice undergoing passive systemic anaphylaxis. Our data suggest that LA may serve as a feedback inhibitor that limits mast cell-mediated inflammation.

Disruption of the suprachiasmatic nucleus blunts a time of day-dependent variation in systemic anaphylactic reaction in mice.

Anaphylaxis is a severe systemic allergic reaction which is rapid in onset and potentially fatal, caused by excessive release of mediators including histamine and cytokines/chemokines from mast cells and basophils upon allergen/IgE stimulation. Increased prevalence of anaphylaxis in industrialized countries requires urgent needs for better understanding of anaphylaxis. However, the pathophysiology of the disease is not fully understood. Here we report that the circadian clock may be an important regulator of anaphylaxis. In mammals, the central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes and entrains peripheral circadian clock present in virtually all cell types via neural and endocrine pathways, thereby driving the daily rhythms in behavior and physiology. We found that mechanical disruption of the SCN resulted in the absence of a time of day-dependent variation in passive systemic anaphylactic (PSA) reaction in mice, associated with loss of daily variations in serum histamine, MCP-1 (CCL2), and IL-6 levels. These results suggest that the central SCN clock controls the time of day-dependent variation in IgE-mediated systemic anaphylactic reaction, which may provide a novel insight into the pathophysiology of anaphylaxis.

A20-deficient mast cells exacerbate inflammatory responses in vivo.

Mast cells are implicated in the pathogenesis of inflammatory and autoimmune diseases. However, this notion based on studies in mast cell-deficient mice is controversial. We therefore established an in vivo model for hyperactive mast cells by specifically ablating the NF-κB negative feedback regulator A20. While A20 deficiency did not affect mast cell degranulation, it resulted in amplified pro-inflammatory responses downstream of IgE/FcεRI, TLRs, IL-1R, and IL-33R. As a consequence house dust mite- and IL-33-driven lung inflammation, late phase cutaneous anaphylaxis, and collagen-induced arthritis were aggravated, in contrast to experimental autoimmune encephalomyelitis and immediate anaphylaxis. Our results provide in vivo evidence that hyperactive mast cells can exacerbate inflammatory disorders and define diseases that might benefit from therapeutic intervention with mast cell function.

Control of microbial attachment by inhibition of ATP and ATP-mediated autoinducer-2.

In this study, 2,4-dinitrophenol (DNP), a typical chemical uncoupler, was employed to investigate the possible roles of ATP and autoinducer-2 (AI-2) of suspended microorganisms in attachment onto nylon membrane and glass slide surfaces. Results showed that DNP could disrupt ATP synthesis, subsequently led to a reduced production of AI-2 which is a common signaling molecule for cellular communication. Attachment of suspended microorganisms exposed to DNP was significantly suppressed as compared to microorganisms without contact with DNP. These suggest that an energized state of suspended microorganisms would favor microbial attachment to both nylon membrane and glass slide surfaces. The extent of microbial attachment was found to be positively related to the AI-2 content of microorganisms. This study offers insights into the control of biofouling by preventing initial microbial attachment through inhibition of energy metabolism.

Anti-allergic effects of Lycopus lucidus on mast cell-mediated allergy model.

The current study characterizes the mechanism by which the aqueous extract of Lycopus lucidus Turcz. (Labiatae) (LAE) decreases mast cell-mediated immediate-type allergic reaction. The immediate-type allergic reaction is involved in many allergic diseases such as asthma and allergic rhinitis. LAE has been used as a traditional medicine in Korea and is known to have an anti-inflammatory effect. However, its specific mechanism of action is still unknown. LAE was anally administered to mice for high and fast absorption. LAE inhibited compound 48/80-induced systemic reactions in mice. LAE decreased the local allergic reaction, passive cutaneous anaphylaxis, activated by anti-dinitrophenyl (DNP) IgE antibody. LAE dose-dependently reduced histamine release from rat peritoneal mast cells activated by compound 48/80 or anti-DNP IgE. Furthermore, LAE decreased the secretion of TNF-alpha and IL-6 in phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187-stimulated human mast cells. The inhibitory effect of LAE on the pro-inflammatory cytokine was p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) dependent. LAE attenuated PMA plus A23187-induced degradation of IkappaBalpha and nuclear translocation of NF-kappaB, and specifically blocked activation of p38 MAPK, but not that of c-jun N-terminal kinase and extracellular signal-regulated kinase. Our findings provide evidence that LAE inhibits mast cell-derived immediate-type allergic reactions and involvement of pro-inflammatory cytokines, p38 MAPK, and NF-kappaB in these effects.

Indirect effects of oral tolerance to ovalbumin interfere with the immune responses triggered by Schistosoma mansoni eggs.

The objective of the present study was to investigate whether the injection of a tolerated protein (indirect effects) affects the formation of granulomas around Schistosoma mansoni eggs trapped in the lungs after intravenous (iv) injection into normal (noninfected) C57BL/6 mice (6 animals per group). To induce oral tolerance to chicken egg ovalbumin a 1/5 dilution of egg white in water was offered ad libitum in a drinking bottle for 3 days. Control mice received water. After 7 days, control and experimental animals were injected iv with 2,000 S. mansoni eggs through a tail vein. In some mice of both groups the iv injection of eggs was immediately followed by intraperitoneal (ip) immunization with 10 micro g of dinitrophenylated conjugates of ovalbumin (DNP-Ova) emulsified in complete Freund's adjuvant (CFA) or only CFA; 18 days later, mice were bled and killed by ether inhalation. The lungs were fixed in formalin and embedded in paraffin. Serial sections of 5 m were stained with Giemsa, Gomori's silver reticulin and Sirius red (pH 10.2). Granuloma diameters were measured in histological sections previously stained with Gomori's reticulin. Anti-DNP and anti-soluble egg antigen (SEA) antibodies were analyzed by ELISA. In mice orally tolerant to ovalbumin the concomitant ip injection of DNP-Ova resulted in significantly lower anti-SEA antibodies (ELISA*: 1395 +/- 352 in non-tolerant and 462 +/- 146 in tolerant mice) and affected granuloma formation around eggs, significantly decreasing granuloma size (area: 22,260 +/- 2478 to 12,993 +/- 3242 m ). Active mechanisms triggered by injection of tolerated antigen (ovalbumin) reduce granuloma formation.

Neurokinin-1 (NK-1) receptor is required in antigen-induced cystitis.

Interstitial cystitis (IC) is a debilitating disease that has been adversely affecting the quality of women's lives for many years. The trigger in IC is not entirely known, and a role for the sensory nerves in its pathogenesis has been suggested. In addition to inflammation, increased mast cell numbers in the detrusor muscle have been reported in a subset of IC patients. Experimentally, several lines of evidence support a central role for substance P and neurokinin-1 (NK-1) receptors in cystitis. The availability of mice genetically deficient in neurokinin-1 receptor (NK-1R(-/-)) allows us to directly evaluate the importance of substance P in cystitis. An unexpected finding of this investigation is that NK-1R(-/-) mice present increased numbers of mast cells in the bladder when compared with wild-type control mice. Despite the increase in mast cell numbers, no concomitant inflammation was observed. In addition, bladder instillation of wild-type mice with a sensitizing antigen induces activation of mast cells and an acute inflammatory response characterized by plasma extravasation, edema, and migration of neutrophils. Antigen-sensitized NK-1R(-/-) mice also exhibit bladder mast cell degranulation in response to antigen challenge. However, NK-1R(-/-) mice are protected from inflammation, failing to present bladder inflammatory cell infiltrate or edema in response to antigen challenge. This work presents the first evidence of participation of NK-1 receptors in cystitis and a mandatory participation of these receptors on the chain of events linking mast cell degranulation and inflammation.

Induction of secretory and serum antibody responses following oral administration of antigen with bioadhesive degradable starch microparticles.

Bioadhesive degradable starch microparticles were used to deliver antigen and immunoglobulin A (IgA)-enhancing cytokines to the oral mucosa. Degradable starch microparticle immunization groups consisted of rats dosed topically at the sublingual epithelium of the oral cavity, by subcutaneous injection in the vicinity of the major salivary glands or by oral intubation with degradable starch microparticles containing dinitrophenyl-bovine serum albumin +/- IL-5/IL-6 +/- penetration enhancer (alpha-lysophosphatidylcholine). Dinitrophenyl-bovine serum albumin was also adsorbed onto alum for salivary gland vicinity injection and administered to the oral cavity in soluble form. Animals were subjected to 3 immunization cycles, and sequential samples were assayed by radioimmunoassay for salivary IgA, tear IgA and serum IgG anti-dinitrophenyl antibodies after secondary and tertiary immunization. Salivary IgA responses were highest in degradable starch microparticle groups receiving penetration enhancer at 71 days post-secondary immunization and continued in one degradable starch microparticle((oral cavity) and two injected (salivary gland vicinity) groups for up to 88 days post-tertiary immunization. Long-term tear responses were also observed in degradable starch microparticle groups receiving penetration enhancer, but they dissipated before the salivary gland-alum responses following tertiary immunization. Serum IgG responses were most pronounced in salivary gland groups, but long-term low level responses were detectable in oral cavity groups receiving degradable starch microparticle formulations with penetration enhancer. Inclusion of IL-5 and IL-6 in oral cavity-delivered degradable starch microparticle formulations consistently enhanced tear IgA while only upregulating salivary IgA antibody responses at early time points post immunization. IL-5 and IL-6 did not enhance serum IgG antibodies in any group. These data indicate that bioadhesive degradable starch microparticles can be used as a vehicle to deliver antigen and cytokine signals to the oral cavity and, when delivered in combination with a penetration enhancer, can potentiate long-term salivary IgA responses.

Virus-specific B-lymphocytes are probably the primary targets for Aleutian disease virus.

368 1- to 5-year-old mink of wild-type or black genetic background were infected with Aleutian disease virus (ADV) naturally or using virus-containing immune complexes or purified virus. Thirty of the mink were immunized with dinitrophenol-conjugated ovalbumin (DNP-OA) before and during infection. Blood samples were taken at monthly intervals. We found that weak (and transient) monoclonal or oligoclonal immunoglobulin components were present in the plasma or serum approximately 1 month after infection, as judged by zone electrophoresis. In a few cases, we found quite stable myeloma-like hypergammaglobulinemia, which usually occurs much later in the infection. All sera with monoclonal immunoglobulin components and most of the sera with immunoglobulins of restricted heterogeneity were analysed by crossed serum line immunoelectrophoresis. In all cases, the distinct immunoglobulins were found to have antibody activity to ADV proteins. In the few sera from DNP-OA-immunized mink showing restricted immunoglobulin heterogeneity, this was also the case. The findings from the study imply that ADV-specific B lymphocytes are probably the primary targets for ADV. The resulting ADV replication introduces a "pseudo-transformation" stage, so that the infected B lymphocytes proliferate and differentiate to an extreme degree. The mechanism behind this B-cell pseudotransformation ability of ADV is a puzzle. It may, however, be important, that the p75/85 structural polypeptides of ADV contain an amino acid sequence almost identical to the GTP-binding pocket of the Ras oncogene.

The behavioral effects of pesticides in male mice.

Male Swiss mice, 25-30 g, were utilized to define some of the behavioral effects of the herbicides Lasso [alachlor 43%; (A)], Basalin [fluchloralin 45%; (F)], Premerge 3 [dinoseb 51%; (D)], and the fungicide Maneb-80 [maneb 80%; (M)]. These compounds were tested for their effects on locomotor activity and for their ability to establish a conditioned taste aversion following oral or dermal exposure. Individual and grouped (N = 5) activity measures were assessed immediately following the dermal administration of the commercially available pesticide formulations. Grouped activity measures were also assessed following the oral administration of the compounds. Total activity was significantly (p less than 0.05) increased over vehicle controls in both grouped and individual subjects by A, F, and D following dermal administration. Grouped activity measures were also increased by A, F, D, and M following the oral administration of the compounds. Similar subjects were tested in a conditioned taste aversion paradigm using a normally preferred 0.3% saccharin solution. Animals were given 30 min access to the saccharin solution followed immediately by the administration of the pesticide or control solution. Twenty-four hours later, animals were given the choice of 2 solutions, one containing water and the other the 0.3% saccharin solution. The percent saccharin consumed and the total fluid intake were calculated for each group (N = 8/group). A, F, and D produced a significant aversion to (N = 8/group) the saccharin following both oral and dermal administration. Oral administration of M, but not dermal exposure, also resulted in a flavor aversion. Total fluid intake, however, was not altered by any of the treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

The specificity of T lymphocyte responses to chemically defined antigens.

A system is described that allows the definition of T cell receptor specificity with some precision. It involves immunization of guinea pigs with hapten coupled to mycobacteria. The T cells of such animals respond to many but not all carriers modified by that hapten. Such T cells recognize neither hapten nor carrier alone, but rather determinants involving both the hapten and the carrier. No evidence for hapten-specific T cells was found. A model of the antigen binding site of the T cell receptor emerged from these experiments. According to this model, the T cell receptor consists of a single site of relatively large extent involving multiple subsites which are of low and roughly equal affinity. Thus, the haptenic group is not immunodominant for T cells as it is for B cells and for anti-hapten antibody. This suggests that the antigen binding receptor on T cells differs in some fundamental way from that on B cells. It is proposed that antigen recognition by T cells is mediated by an immunoglobulin heavy chain variable region that is not paired with an immunoglobulin light chain variable region.

Inhibition of thyroxine binding to adenohypophysial proteins in vitro by 2,4-dinitrophenol administered in vivo.

Male and female rats were given oestradiol benzoate (1 mg as a microcrystal aqueous suspension i.m. twice a week), 0.0033% 2.4-dinitrophenol (DNP) in their food (about 1 mg/rat/day), or 0.1% DNP in their food (about 30 mg/rat/day), or both oestradiol and DNP. The smaller DNP dose mildly stimulated food consumption and did not affect body weight. The larger dose strongly inhibited food consumption in the first two weeks of the experiment; consumption then returned to the control level, but body weight fell markedly at the same time. After 3 weeks' administration of both the small and the large dose of DNP, adrenal weight in the males was raised and the weight of the gonads was unchanged. The large DNP dose severely reduced the weight of the seminal vesicles and the uteri. It also inhibited the accumulation of radioiodine in the thyroid of both males and females. Isolated administration of the oestrogen raised adrenal weight in the males and ovarian and uterine weight in the females; it reduced the weight of the testes and seminal vesicles. These reactions were not affected by DNP. A pronounced oestradiol-induced increase in the weight of the adenohypophyses was accompanied by raised thyroxine binding to the adenohypophysial proteins in vitro. DNP inhibited the growth reaction of the adenohypophysis to the oestrogen only slightly and non-significantly, but significantly inhibited the thyroxine binding reaction to the adenohypophysial proteins in vitro. By itself, DNP had no effect on adenohypophysial weight, but reduced thyroxine binding to the adenohypophysial proteins in vitro, especially in males. The effect of DNP was similar to that of thyroxine observed in earlier experiments; nothing is known of its mechanism.