Background: Circulating autoantibodies against FcϵRI, IgE, or both occur in approximately one third of patients with chronic idiopathic urticaria (CIU), but not all autoantibodies initiate histamine release. Objective: We sought to classify patients with CIU into subsets on the basis of serum bioactivity and immunoreactivity and to examine the relationship between newly defined subtype and disease severity. Methods: Sera from patients with CIU (n = 78), dermog-raphism (n = 15), and cholinergic urticaria (n = 10) and sera from healthy subjects (n = 39) were analyzed by means of Western blot analysis for anti-FcϵRI autoantibodies and for histamine release from basophils and dermal mast cells. In vivo reactivity of autologous serum was tested by means of intradermal injection, and CIU severity was determined on the basis of clinical interview. Results: We classified sera from patients with CIU into 5 subsets: immunoreactive histamine-releasing anti-FcϵRI autoantibodies (n = 20 [26%]); immunoreactive anti-FcϵRI autoantibodies without histamine-releasing activity (n = 12 [15%]); anti-IgE-like autoantibodies (n = 7 [9%]); serum containing a mast cell-specific histamine-releasing factor (n = 7 [9%]); and sera with no identifiable factor (n = 32 [41%]). Patients with serum histamine-releasing activity had more severe urticaria than patients without such activity. Positive skin test responses to autologous sera were associated with histamine-releasing anti-FcϵRI autoantibodies but not with non-histamine-releasing anti-FcϵRI autoantibodies. Neither healthy subjects nor patients with dermographism or cholinergic urticaria had his-tamine-releasing anti-FcϵRI autoantibodies. Conclusion: These data support the specificity of functional anti-FcϵRI autoantibodies to CIU. The identification of distinctive subsets of patients suggests that other pathogenic mechanisms occur in CIU in addition to direct ligation of FcϵRI by autoantibodies causing dermal mast cell degranulation. Elucidating these mechanisms might lead to new treatments for CIU. (J Allergy Clin Immunol 2002;110:492-9.)

The major histocompatibility complex (MHC) acts as a marker for self during T-cell ontogeny and is associated with the pathogenesis of many autoimmune diseases, Recent investigations have shown about 30% of patients with chronic idiopathic urticaria (CIU) have IgG autoantibodies against the high-affinity IgE receptor, Fc epsilon RI, or IgE, A link between MHC class II alleles and CIU has not been reported previously, DNA was extracted from blood of 100 Caucasian patients with CIU, and the MHC class II type determined using the polymerase chain reaction with sequence-specific primers, testing for DRB and DQB1 alleles. The frequency of alleles in CIU patients was compared with that found in 603 controls. Further human leucocyte antigen (HLA) typing on patient subsets, classified by the patients' responses to intradermal injection of autologous serum and their serum-induced histamine release from basophil leucocytes of healthy donors, was undertaken. HLA DRB1*04 (DR4) and its associated allele, DQB1*0302 (DQ8), are raised in CIU patients compared with a control population (P = 2 x 10(-5) and P = 2 x 10(-4), respectively). HLA DRB1*15 (DR15) and its associated allele, DQB1*06 (DQ6), are significantly less frequently associated with CIU, The HLA DRB1*04 association is particularly strong (corrected P = 3.6 x 10(-6)) for patients whose serum has in vivo and in vitro histamine-releasing activity, HLA class II typing is consistent with the concept that CIU is a heterogeneous disease, and supports an autoimmune pathogenesis in a subset of patients.

One-third of patients with chronic idiopathic urticaria (CIU) have circulating functional autoantibodies against the high affinity IgE receptor FcepsilonRI, or IgE. The intradermal injection of autologous serum causes a weal and flare reaction in many patients with CIU, and this reaction forms the basis of the autologous serum skin test (ASST). We have determined the parameters of the ASST which define the optimal sensitivity and specificity for the identification of patients with autoantibodies. Two physicians (R.A. S. and C.E.H.G.) performed ASSTs in a total of 155 patients with CIU, 40 healthy control subjects, 15 patients with dermographism, nine with cholinergic urticaria and 10 with atopic eczema. Patients were classified as having functional autoantibodies by demonstrating in vitro serum-evoked histamine release from the basophils of two healthy donors. There were significant differences (P < 0.001) in the mean weal diameter, weal volume, weal redness and flare area of the intradermal serum-induced cutaneous responses at 30 min between patients with CIU with autoantibodies and either those without autoantibodies or control subjects. The optimum combined sensitivity and specificity of the ASST was obtained if a positive test was defined as a red serum-induced weal with a diameter of >/= 1.5 mm than the saline-induced response at 30 min. For R.A.S. and C.E.H.G., the ASST sensitivity was 65% and 71% and specificity was 81% and 78%, respectively. Using these criteria, the following subjects had positive ASSTs: none of 15 dermographics, none of 10 atopics, one of nine cholinergics and one of 40 controls.

Background: Previous studies defining the clinical features of patients with chronic idiopathic urticaria (CIU) were performed before the identification of functional autoantibodies against Fc epsilon RI and/or IgE, now known to be present in approximately 30% of patients with CIU.Objective: Our purpose was to determine whether there are differences between patients with and those without autoantibodies in the clinical features or severity of CIU.Methods: The clinical features of 107 patients with CIU were evaluated prospectively. Patients were identified as having functional autoantibodies on the basis of the serum-evoked histamine release in vitro from the basophils of 2 healthy donors.Results: Patients with autoantibodies (31%) had more wheals (P =.005), a wider distribution of wheals (P =.009), higher itch scores for the most severe episodes of itching (P =.002), more systemic symptoms (P =.03), and lower serum IgE levels (P <.0005) than patients without autoantibodies.Conclusion: The presence of autoantibodies indicates a subset of patients with more severe CIU.

Cytokines induced in skin by ultraviolet radiation cause local and systemic immunosuppression, Tumor necrosis factor alpha, interleukin-l, and interleukin-10 are key mediators in the mouse, but less is known about cytokine synthesis and function in ultraviolet-irradiate. human skin. We exposed human skill to 3 minimal erythema doses of solar-simulated radiation and raised suction blisters at intervals to 72 h. Alloantigen presentation was suppressed in a mixed epidermal cell-lymphocyte reaction by 69% from 4 to 15 h post-solar-simulated radiation, but recovered to control values by 24 h. Tumor necrosis factor alpha was raised at 4 h after solar-simulated radiation, reached a maximum 8-fold increase at 15 h, then rapidly declined to control values. Interleukin-1 alpha and interleukin-1 beta were first increased at 15 h, and remained raised to 72 h, although interleukin-1 beta declined from its 15 h maximum, Interleukin-10 increased a maximum 2-fold between 15 and 24 h, coincident with recovery of mixed epi-dermal cell-lymphocyte reaction responses and downregulation of tumor necrosis factor alpha and interleukin-1 beta Solar-simulated radiation differentially affected soluble tumor necrosis factor alpha receptors; soluble tumor necrosis factor-RI was suppressed 33% at 8-15 h whereas soluble tumor necrosis factor-RII increased 2-fold from 15 to 48 h. Interleukin-1 receptor antagonist was raised at all times post-irradiation. Interleukin-12 was not detectable in control or irradiated skin. These kinetics suggest the tumor necrosis factor alpha network has primary importance in ultraviolet-damaged human skin, The small increase in interleukin-10 implies that 3 minimal erythema doses of solar-simulated radiation is the threshold dose for its induction and local, rather than systemic, functions for interleukin-10 in immunosuppression and regulation of other cytokines.

Stimulation of human epidermal keratinocytes with irritant phenolic compounds leads to the release of potent immunologically active mediators. A principal protein component released into the cell supernatant has been shown to have the biological and immunological properties appropriate for the primary cytokine interleukin-1-alpha. Electrospray mass spectrometry (ESI-MS) has been used to show that the mass of this protein corresponds to only a part of the published cDNA based sequence. ESI-MS has also been used to describe the secondary, immunologically active components, formed when fibroblasts are incubated with an extract of this protein.

BACKGROUND:Peripheral blood basophils are reduced in some chronic urticaria patients when counted with granule stains. Approximately 30% of patients with severe chronic urticaria have functional autoantibodies which release histamine from healthy donor basophils in vitro but the relationship between basophil numbers in vivo and serum histamine releasing activity has not been studied. OBJECTIVE:To determine the relationship between basophil numbers and serum basophil histamine releasing activity and to assess whether basophils are present, but undetectable, in peripheral blood with granule stains by using a new flow cytometric method based on surface immunophenotype. METHODS:Basophils were counted manually by a chamber method using a granule stain and by flow cytometry using dual staining with anti-IgE and anti-Fc epsilonRI in 25 chronic idiopathic urticaria patients and 25 healthy controls. Serum histamine releasing activity was assessed on healthy donor basophils in vitro and by the weal response to autologous serum skin testing in vivo (patients only). RESULTS:Basophils were significantly reduced in chronic urticaria by manual counting and flow cytometry. A subgroup of seven patients with in vitro histamine releasing activity showed a marked reduction or absence of basophils by both methods. There were no obvious distinguishing clinical characteristics between these patients and the others; six of them showed positive autologous serum skin-test responses. On comparing the two methods, the manual basophil counts were generally lower than flow cytometric counts. Agreement over the full range of values was not strong and therefore counts obtained by the two methods are not directly interchangeable. CONCLUSION:Basopenia in chronic idiopathic urticaria is associated with serum basophil histamine releasing activity in a subgroup of patients. The lack of granule and surface immunophenotype staining suggests a reduction in numbers rather than an inability to detect circulating degranulated cells by conventional counting methods.

Previous studies identified autoantibodies against the IgE high affinity receptor alpha-chain, Fc epsilon RI alpha, in sera of selected patients with severe chronic idiopathic urticaria. We have now determined the incidence of anti-Fc epsilon RI alpha autoantibodies in a group of 163 patients. Intradermal injection of autologous serum caused skin reactions indicative of mast cell degranulation in 98 (60%) patients. Based on histamine release from IgE-sensitized and nonsensitized basophil leukocytes of healthy donors, we detected anti-Fc epsilon RI alpha autoantibodies in sera from 38 (23%) urticaria patients and evidence for anti-IgE antibodies in a further nine patients. The sera that released histamine from basophils induced histamine release (4-34%, n = 12) from mast cells in incubated skin slices. Protein-G affinity chromatography of sera demonstrated that mast cell histamine release was IgG-mediated. Preincubation of sera or the IgG fraction with a recombinant alpha-chain of Fc epsilon RI inhibited histamine release from mast cells and basophils. Further studies with the mouse anti-human Fc epsilon RI alpha antibody 29C6 showed that mast cells and basophils were similarly sensitive to IgG-mediated direct cross-linking of Fc epsilon RI, with 0.01-1.0 micrograms/ml 29C6 evoking histamine release in each case. These studies demonstrate that circulating levels of anti-Fc epsilon RI alpha autoantibodies mediate histamine release from skin mast cells in vitro and, taken together with in vivo evidence of mast cell degranulation following intradermal injection of autologous serum, support the concept that anti-Fc epsilon RI alpha autoantibodies are relevant to the pathogenesis of severe chronic urticaria in about 25% of patients.

The extent of PAF synthesis and its role in inflammatory skin disorders are not known. Skin exudates were collected from suction blisters or bbrasions on healthy and inflamed human skin and analysed by gc-ms. The mean PAF andlyso-PAF values for skin of healthy subjects were 1.86±0.92 (n=7) and 160±21 (n=8) nM, respectively, in suction blister exudates, and 0.38±0.08 (n=14) and 11.0±1.9 (n=13) pmol/abrasion, respectively. PAF levels were not altered in psoriasis, nickel contact dermatitis, delayed pressure urticaria, mastocytosis or immediate and late phase reactions to antigen. Significantly, lesslyso-PAF was found in uninvolved skin of psoriasis. Increasedlyso-PAF was found in nickel contact dermatitis compared with untreated skin of the same subjects or skin or normal healthy volunteers. We conclude that, if PAF is a significant mediator of inflammation in human skin, it may remain associated with the synthesising cell or act in its immediate proximity.

Electrospray mass spectrometry has been employed to detect and characterize the members of a closely related family of immunologically active proteins (chemokines) produced in cell culture by stimulated fibroblasts. The reliability of the method to produce precise data concerning the relative proportions of proteins present in mixtures was investigated in a model system and found to be satisfactory in a range of 10:1 in concentration.

Previous studies have shown that scale from lesional psoriatic skin contains substantial amounts of platelet activating factor (PAF). In this study, PAF and its immediate precursor, lyso-PAF, were measured in exudates from abrasions on lesional and uninvolved psoriatic skin, and from skin of healthy subjects. The mean amounts of PAF recovered from lesional and uninvolved psoriatic skin (n = 13) and from healthy skin (n = 14) were not significantly different (range 0.05-2.14 pmol/sample). Mean recoveries of lyso-PAF from lesional psoriatic skin (n = 9) and skin of healthy subjects (n = 13) were also similar (9.5 +/- 1.9 and 11.0 +/- 1.9 pmol/sample, respectively), but significantly less lyso-PAF was found in exudates from the uninvolved psoriatic skin (n = 9; 3.1 +/- 0.4 pmol/sample; P < 0.01 relative to both lesional psoriasis and healthy skin). The finding of reduced lyso-PAF in uninvolved psoriatic skin was unexpected because increased phospholipase-A2 activity is associated with psoriasis. These results do not support the hypothesis that extracellular PAF contributes significantly to the inflammation associated with psoriasis.