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.

A novel model of IgE-mediated passive pulmonary anaphylaxis in rats.

Mast cells are central effector cells in allergic asthma and are augmented in the airways of asthma patients. Attenuating mast cell degranulation and with it the early asthmatic response is an important intervention point to inhibit bronchoconstriction, plasma exudation and tissue oedema formation. To validate the efficacy of novel pharmacological interventions, appropriate and practicable in vivo models reflecting mast cell-dependent mechanisms in the lung, are missing. Thus, we developed a novel model of passive pulmonary anaphylaxis in rats. Rats were passively sensitized by concurrent intratracheal and intradermal (ear) application of an anti-DNP IgE antibody. Intravenous application of the antigen, DNP-BSA in combination with Evans blue dye, led to mast cell degranulation in both tissues. Quantification of mast cell degranulation in the lung was determined by (1) mediator release into bronchoalveolar lavage, (2) extravasation of Evans blue dye into tracheal and bronchial lung tissue and (3) invasive measurement of antigen-induced bronchoconstriction. Quantification of mast cell degranulation in the ear was determined by extravasation of Evans blue dye into ear tissue. We pharmacologically validated our model using the SYK inhibitor Fostamatinib, the H1-receptor antagonist Desloratadine, the mast cell stabilizer disodium cromoglycate (DSCG) and the ß2-adrenergic receptor agonist Formoterol. Fostamatinib was equally efficacious in lung and ear. Desloratadine effectively inhibited bronchoconstriction and ear vascular leakage, but was less effective against pulmonary vascular leakage, perhaps reflecting the differing roles for histamine receptor sub-types. DSCG attenuated both vascular leakage in the lung and bronchoconstriction, but with a very short duration of action. As an inhaled approach, Formoterol was more effective in the lung than in the ear. This model of passive pulmonary anaphylaxis provides a tissue relevant readout of early mast cell activity and pharmacological benchmarking broadly reflects responses observed in patients with asthma.

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.

Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar).

This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens - dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) - administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.

Induction of immunological tolerance in chickens inoculated with xenogeneic antigens at a late stage of embryonic development.

To induce immunotolerance, chicken embryos were inoculated with BSA on embryonic incubation day (EID) 20. All hatched chickens were challenged with BSA four times at 10-day intervals, beginning at 3 weeks of age. Serum anti-BSA antibodies were analyzed and our results show that immunotolerance was obtained in the group micro-injected with 1 mg of BSA. We administered BSA labeled with 2,4-dinitrophenyl (DNP) into blood vessels on EID 20 and detected the distribution of BSA by immunohistochemistry. Our results show that DNP-BSA was located in the bursal cortex, thymus and spleen. PBMCs were separated on the seventh day after the fourth immunization to determine the effect of treatments on lymphocytes and the percentage of CD4(+) T cells among the CD3(+) T cell population. Results from these experiments show that there was an influence on T lymphocyte proliferation, with the number of CD4(+) T cells in the tolerance group significantly increased. To determine whether tolerance was induced in B cells, 2,4,6-trinitrophenyl coupled to BSA (TNP-BSA) was inoculated into birds, followed by detection of anti-TNP antibodies in the serum. Immunological tolerance in B cells was not observed following these experiments. The results from our study demonstrate that immunological tolerance was induced in T cells when 1 mg of BSA was micro-injected on EID 20.

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.

IgE cross-linking or lipopolysaccharide treatment induces recruitment of Th2 cells to the lung in the absence of specific antigen.

We previously showed that Th1 cells can increase recruitment of Th2 cells to the lungs even in the absence of the Th2-specific Ag. The fact that Th2 recruitment is independent from the Th2 cell Ag suggested that Th1 cells may support Th2 cell recruitment using their Ag-nonspecific proinflammatory functions. To investigate the potential for inflammatory stimuli that are distinct from Ag-specific signals to affect the recruitment of T cells, we tested whether cross-linking of IgE or treatment with LPS modulated influx of Th2 cells into the airways in the presence or absence of inhaled Ag. When naive mice that had been treated with OVA-specific Th2 cells and passively sensitized with anti-DNP IgE were challenged by intranasal administration of either DNP-haptenated OVA or DNP-BSA, increased numbers of Th2 cells were recruited to the lung compared with mice challenged intranasally with OVA alone. Intranasal administration of LPS also increased recruitment of Th2 cells to the airways. These two distinct inflammatory stimuli increased the numbers of recruited Th2 cells equally with or without concurrent challenge using the cognate Th2 Ag. This Ag-independent recruitment of Th2 cells to the lung was not associated with localization of these cells to the regional lymph nodes and was independent of Th2 cell activation. Interestingly, P- or E-selectin contributed to Th2 cell recruitment to the lung. These data suggest that Th2 cells of the adaptive immune response are similar to cells of the innate immune response in their lack of requirement for protein Ag to initiate cell recruitment. They demonstrate further that recruitment can occur independently of Ag-dependent activation.

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.

Enhancement of rat tear IgA antibody responses following intranasal immunization with antigen and CpG ODN.

PURPOSE: To determine the effect of unmethylated oligodeoxynucleotides containing bacterial CpG motifs (CpG ODN) on the induction of rat tear IgA antibody responses. METHODS: Rats were immunized intranasally with either soluble dinitrophenylated bovine serum albumin (DNP-BSA) or poly(lactide-co-glycolide) (PLG) encapsulated DNP-BSA in combination with CpG ODN. The animals received two immunizations 21 days apart. Following the second immunization, tear, saliva and serum samples were collected for 28 days and analyzed for antigen specific antibodies. Tear IgA, saliva IgA and serum IgG antibody concentrations were determined by ELISA. RESULTS: Co-administration of CpG ODN with either soluble or encapsulated antigen resulted in significantly elevated levels of both tear and salivary IgA antibodies as well as levels of serum IgG antibodies. Microencapsulated DNP-BSA plus CpG ODN elicited higher levels of IgA antibodies in tears than did soluble antigen plus CpG ODN. CONCLUSIONS: CpG ODN is an effective mucosal immune modulator for enhancing rat tear IgA antibody responses to both soluble and microencapsulated antigens.

Role of the IgE-mediated system in eosinophil recruitment triggered by two consecutive cycles of sensitisation and challenge in rats.

In this study, we postulated that repeated cycles of IgE passive sensitisation and antigen challenge may play a role in up-regulating eosinophil response in allergic conditions. Antigen-mediated stimulation of the pleural cavity of rats passively sensitised with a single injection of IgE anti-DNP resulted in mast cell degranulation, increase in vascular permeability and mild neutrophilia, but no pleural eosinophilia. In contrast, a second cycle of sensitisation and challenge, performed within 7 days, showed a marked eosinophilia in parallel with a lower plasma leakage and comparable neutrophilia. The eosinophilic phenomenon was not reproduced when (1) IgE sensitisation or antigen challenge was omitted in the first cycle, or (2) the first cycle was replaced by either a histamine and 5-HT dual challenge or a PAF challenge. Furthermore, we found an increase in eotaxin levels in animals subjected to two rather than one cycle of sensitisation and challenge. Treatment with the PAF receptor antagonist BN 52021 or with the lipoxygenase inhibitor zileuton, but not mast cell granule depletion, prevented the allergen-evoked eosinophil accumulation in rechallenged animals. Our results indicate that repeated cycles of IgE-driven inflammation may lead to eosinophil accumulation in a mechanism dependent on eotaxin, PAF and leukotrienes.

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.

Antigen-specific T cell tolerance down-regulates mast cell responses in vivo.

Fel d I is the major cat allergen that induces asthma and allergic rhinitis in humans. To investigate the mechanism of allergic responses to this allergen, a mouse model was developed. Mice sensitized to chain 1 of Fel d I exhibited T cell responses, B cell responses, and mast cell responses when challenged with the protein. Subcutaneous injections of peptides containing the dominant T cell epitopes of the allergen induced T cell tolerance in presensitized mice. When challenged with the allergen intratracheally, these tolerized mice produced a decreased amount of histamine in vivo. The decrease in histamine release was not solely dependent on the reduction of allergen-specific IgE. These data show that mast cell activity in mice with an ongoing sensitivity to allergen can be regulated through peptide-induced T cell tolerance.

IL-12 is a potent neonatal vaccine adjuvant.

Neonatal animals show generally poor responsiveness to foreign antigens and are known to display polarized expression of Th2-like cytokines and antibody responses. We now report that newborn mice display a reduction in peripheral expression of the Th1-inducing cytokine, IL-12. Attempts to overcome this decrease by immunization and treatment with IL-12 within 24 h of birth resulted in elevated levels of IFN-gamma and IL-10 mRNA in the spleens of mice compared to animals exposed to antigen only. Moreover, such animals showed dramatic enhancement of IgG2a and IgG2b antibody levels upon adult challenge compared to mice primed with antigen alone. These effects appeared to be due to induction of neonatal B cell memory. IgG1 antibody levels, a measure of Th2 activity, were unaffected or even somewhat enhanced by neonatal IL-12 treatment. Taken together, these results provide evidence that IL-12 administration induces a Th1-like cytokine response in newborns and causes priming for heightened memory antibody responses in vivo. Our findings suggest the use of IL-12 as a vaccine adjuvant in neonates for inducing protection against common childhood pathogens.

Priming or tolerization of the B- and Th2-dependent immune response by the oral administration of OVA-DNP is determined by the antigen dosage.

In the present report we established antigen dosages that induce oral tolerance of Th1 and Th2 lymphocytes or instead prime B- and Th2-dependent immune response and induce the tolerance of Th1 lymphocytes. Using different hapten-carrier systems, we found that low doses of OVA-DNP administered orally primed B and Th2 cells. On the other hand, no priming of B or Th2 cells was found in high-dose-OVA-DNP-fed rats. Low-dose-OVA-DNP-fed rats showed a strong mucosal immune response, with a high number of IgA anti-DNP antibody-forming cells in the lamina propria, while no mucosal immune response was observed in high-dose-OVA-DNP-fed rats. Thirty days after the immunization, tolerization of Th1 lymphocytes was confirmed in low- and high-dose-OVA-DNP-fed rats by diminished antigen-specific proliferation in vitro, reduced titers of anti-DNP IgG2a in serum, reduced expression of CD25 and CD134 molecules in cultured cells exposed to the antigen, reduced DTH reaction, and reduced IL-2 synthesis in culture. On the other hand, a high dose of OVA-DNP led to Th1 and Th2 tolerance, with an inhibition of specific IgG1 and IgG2a anti-DNP antibodies in serum after a parenteral challenge with OVA in CFA. This functional evidence was supported by the direct examination of IL-2 and IL-4 production. Furthermore, whereas in vitro assays seem to indicate that active suppression could be the responsible for Th1 tolerization in low-dose-OVA-DNP-fed rats, the results obtained after the transference of spleen or MLN cells to naive recipients support the idea that a subtractive mechanism is behind the tolerization of Th1 lymphocytes.

Production and characterisation of polyclonal antisera against feline IgE.

Cats, naturally or experimentally infected with Toxocara cati were immunised with dinitrophenylated ascaris antigen (DNP-Asc). All cats developed immediate skin reactivity to DNP coupled to bovine serum albumin (DNP-BSA) and the sera of the nine cats had a heat labile homocytotropic antibody detectable by homologous Prausnitz-Küstner (PK) tests. Reagin-rich fractions were prepared from these sera and used for the preparation of polyclonal antisera in rabbits. Resultant antisera were passed through a immunoabsorbent column of Sepharose 4B coupled to heated normal cat serum. An immunoabsorbent column prepared with the resultant antisera removed the PK reactivity from the cat sera, and the activity was recovered following acid elution. The antiserum failed to detect any recognised immunoglobulin in cat sera, but precipitated with a heat labile protein with gamma-1 electrophoretic mobility in the sera of parasited cats. These findings support the contention that the antisera are specific for feline IgE.

Inductive pathways leading to rat tear IgA antibody responses.

PURPOSE: To define the inductive pathways leading to rat tear IgA antibody responses. METHODS: Fluoresceinated dinitrophenylated bovine serum albumin was encapsulated in poly(lactide-co-glycolide) microparticles and was administered by intranasal, ocular topical, or gastrointestinal routes. Histologic methods were used to determine the microparticles' ability to access tissues associated with mucosal inductive pathways. Rats were immunized with microencapsulated antigen by intranasal or ocular topical routes. Tear IgA and serum IgG antibody concentrations were assessed by radioimmunoassay. The frequency of antibody-secreting cells in tissues, postulated to function in tear IgA induction, was measured by enzyme-linked immunospot assay. RESULTS: Although uptake of microencapsulated antigen was greatest at the site of delivery, ocular topical administration resulted in antigen uptake in the conjunctiva and in nasal-associated lymphoid tissue. Intranasal immunization resulted in earlier and significantly higher tear IgA and serum IgG antibody responses and in higher frequencies of antibody-secreting cells in corresponding draining cervical lymph nodes and lacrimal glands than did ocular topical immunization. CONCLUSIONS: Nasal-associated lymphoid tissue functions as a primary inductive site for tear IgA antibody responses by contributing triggered IgA-committed B cells to the lacrimal gland.

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.

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens.

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response.