Mechanism of topical glucocorticoid treatment of hay fever: IL-5 and eosinophil activation during natural allergen exposure are suppressed, but IL-4, IL-6, and IgE antibody production are unaffected.

BACKGROUND: Allergic rhinitis is traditionally defined as an IgE- and mast cell-mediated hypersensitivity reaction. Allergen challenge models suggest that cytokines and eosinophil mediators may also play roles. However, the causal relationship among inflammatory cells, their products, and patients' symptoms during natural allergen exposure has not been established. OBJECTIVE: We sought to elucidate the mechanisms of seasonal allergic rhinitis and the beneficial effects of topical glucocorticoids. METHODS: Thirty patients with ragweed-induced hay fever and a strongly positive serologic test response for ragweed IgE antibody received budesonide nasal spray or placebo in a randomized, parallel, double-blind study. Nasal wash fluids and sera were collected before and during the hay fever season. The levels of inflammatory mediators and allergen-specific immunoglobulins were measured by immunoassay. The activation markers on blood eosinophils were quantitated by flow cytometry. RESULTS: Compared with placebo-treated patients, budesonide-treated patients had strikingly reduced symptoms. In the placebo group, nasal symptoms correlated with nasal lavage fluid eosinophil-derived neurotoxin and IL-5 levels. At the season peak, the budesonide-treated group had significantly lower nasal fluid eosinophil-derived neurotoxin, IL-5, and soluble intracellular adhesion molecule-1 levels. In the treated group eosinophil expression of CD11b was suppressed at the season peak. In contrast, levels of IL-4 and IL-6 in nasal fluid and the seasonal increases in serum ragweed-specific IgE and nasal fluid IgA antibodies did not differ between groups. CONCLUSION: Eosinophilic inflammation plays a critical role in seasonal allergic rhinitis symptoms. One of the therapeutic effects of glucocorticoids is to suppress this inflammation.

Eosinophil granule proteins in peripheral blood granulocytes.

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (mean +/- SEM) in ng/10(6) eosinophils (and in nM/10(6) eosinophils) were: MBP, 8,982 +/- 611 (641.6); EDN, 3,283 +/- 116 (178.4); ECP, 5,269 +/- 283 (250.9); and EPO, 12,174 +/- 859 (171.5). Basophils from a normal person contained (in ng/10(6) cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/10(6) cells) MBP, 3 +/- 0.5; EDN, 72 +/- 9; and ECP, 50 +/- 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

Participation of eosinophils in the toxic oil syndrome.

The participation of eosinophils in the Spanish toxic oil syndrome (TOS) was investigated. Eosinophil infiltration and degranulation in tissues from 52 patients with the TOS were examined by immunofluorescence staining for the eosinophil granule major basic protein (MBP). Serum MBP levels were determined in sera from 323 patients. Eosinophil infiltration and degranulation were found in several tissues, especially during the acute phase of the TOS, and serum MBP was significantly elevated during all phases of the disease, suggesting that eosinophils play a role in the pathogenesis of the TOS.

An investigation of the cause of the eosinophilia-myalgia syndrome associated with tryptophan use.

BACKGROUND: The eosinophilia-myalgia syndrome is a newly recognized illness that has been associated with the consumption of tryptophan products. It is not known whether the cause is related to the tryptophan itself or to chemical constituents introduced by the manufacturing process. METHODS: To describe the epidemiology of the eosinophilia-myalgia syndrome further and elucidate a possible association with the manufacturing process, we conducted surveillance for the syndrome in Minnesota, a community survey of tryptophan use in Minneapolis-St. Paul, and a case-control study to assess potential risk factors, including the use of tryptophan from different manufacturers. We performed high-performance liquid chromatography on tryptophan samples to identify other chemical constituents. RESULTS: The prevalence of tryptophan use increased from 1980 to 1989 and was highest among women. Among the subjects for whom the source of the tryptophan was known, 29 of 30 case patients (97 percent) and 21 of 35 controls (60 percent) had consumed tryptophan manufactured by a single company (odds ratio, 19.3; 95 percent confidence interval, 2.5 to 844.9; P less than 0.001). This company used a fermentation process involving Bacillus amyloliquefaciens to manufacture tryptophan. Analysis of the manufacturing conditions according to the retail lot demonstrated an association between lots used by case patients and the use of reduced quantities of powdered carbon in a purification step (odds ratio, 9.0; 95 percent confidence interval, 1.1 to 84.6; P = 0.014), as well as the use of a new strain of B. amyloliquefaciens (Strain V) (odds ratio, 6.0; 95 percent confidence interval, 0.8 to 51.8; P = 0.04). There was a significant correlation (r = 0.78, P less than 0.001) between the reduced amount of powdered carbon used during manufacturing and the use of the new bacterial strain. High-performance liquid chromatography of this company's tryptophan demonstrated one absorbance peak (peak E) that was present in 9 of the 12 retail lots (75 percent) used by patients and 3 of 11 lots (27 percent) used by controls (odds ratio, 8.0; 95 percent confidence interval, 0.9 to 76.6; P = 0.022). CONCLUSIONS: The outbreak of the eosinophilia-myalgia syndrome in 1989 resulted from the ingestion of a chemical constituent that was associated with specific tryptophan-manufacturing conditions at one company. The chemical constituent represented by peak E may contribute to the pathogenesis of the eosinophilia-myalgia syndrome, or it may be a surrogate for another chemical that induces the syndrome.

Comparison of antifading agents used in immunofluorescence.

Various chemicals including p-phenylenediamine, n-propyl gallate, 1,4-diazobicyclo[2,2,2]-octane and Citifluor were tested to determine their effectiveness in retarding the fading of fluorescein-conjugated antibody bound to eosinophil granule major basic protein in tissues. Immunofluorescence fading and intensity were measured quantitatively using the Zeiss Zonax microphotometer system. All of these agents effectively retarded fluorescence fading; p-phenylenediamine and n-propyl gallate were the most effective. Comparison of glycerol-based and polyvinyl alcohol-based media containing n-propyl gallate or p-phenylenediamine showed that the glycerol-based medium yields higher mean fluorescence intensities than the polyvinyl alcohol-based medium. Slides mounted with p-phenylenediamine-glycerol could be stored for up to 2 weeks without loss of fluorescence intensity. Although p-phenylenediamine has many undesirable chemical properties, we conclude that it is the agent of choice for retarding the fading of tissue preparations stained with fluorescein isothiocyanate conjugates when used in a glycerol-based medium.

Ultrastructural study of eosinophils from patients with the hypereosinophilic syndrome: a morphological basis of hypodense eosinophils.

We investigated the ultrastructural characteristics and the granule major basic protein (MBP) content of hypodense eosinophils from patients with the hypereosinophilic syndrome who had at least 90% hypodense eosinophils in their peripheral blood and compared these cells to normodense eosinophils from normal persons. The hypodense cells (density less than 1.082) contained significantly less MBP than normodense (density greater than 1.082) eosinophils (P less than .001) as measured by radioimmunoassay (RIA). Electron microscopic examination demonstrated a mean of 25.0 +/- 4.4 (X +/- 1 SD) granules per hypodense cell, compared to 30.6 +/- 8.4 granules per cell in the normodense group (P less than .1). The most striking difference between the hypodense and normodense eosinophils was the small individual granule size (X = .14 +/- .05 v .26 +/- .05 micron 2, respectively, P less than .001), and the smaller total granule area (3.2 +/- 1.8 vs 7.7 +/- 3.1 micron 2, respectively, P less than .001). Because the cytoplasmic areas were similar in the two groups, the mean percent area of cytoplasm occupied by granules was significantly lower in the hypodense group (P less than .001). The finding of consistently smaller granules in the presence of equal or fewer granules per cell in the hypodense eosinophils may explain the lower MBP content and thus provide a morphologic basis for the low density of eosinophils in patients with the hypereosinophilic syndrome.

Detection of antibodies to proteases used in laundry detergents by the radioallergosorbent test.

Two proteases, Esperase and Alcalase, derived from Bacillus licheniformis and B. subtilis, respectively, are used in laundry products. In testing for the prevalence of IgE antibodies to these enzymes in sera among 300 laundry product workers, we experienced two problems in the establishment of a reliable RAST for these antigens. The first problem was the propensity of the allergen, Esperase, to undergo autolysis, suggesting that solid-phase Esperase might also lose reactivity through degradation. Treatment of Esperase with phenylmethylsulfonyl fluoride stabilized the enzyme and permitted the synthesis of a stable solid-phase antigen. The second problem was the finding that sera reactive with Esperase in the RAST were also reactive with Savinase, an enzyme from B. licheniformis to which the workers were not exposed. Immunochemical analyses of the three enzymes with specific rabbit antisera by gel diffusion and by two-site immunoradiometric assay demonstrated that they were not cross contaminated to any appreciable extent. RAST inhibition demonstrated that solid-phase Esperase possessed unique allergenic determinants in that the reactivity of IgE antibodies was inhibited by low concentrations of Esperase and only by very high concentrations of Alcalase and Savinase. In contrast, the reactivity of solid-phase Alcalase was occasionally inhibited equally well by Esperase and Alcalase. Most strikingly, the reaction of IgE antibodies with solid-phase Savinase was always inhibited by comparable quantities of Esperase, Alcalase, and Savinase. Thus, the establishment of the RAST for these proteases appears to require the use of phenylmethylsulfonyl fluoride to retard autolysis, and the results must be interpreted with caution because IgE antibodies in certain sera demonstrate cross-reactivity with Alcalase and Savinase.

Microbiologic analyses of nasal polyp tissue.

Nasal polyps from 40 patients were cultured within 2 1/2 hours after surgical removal to determine whether microorganisms were present. The first 20 polyps were cultured for aerobic and anaerobic bacteria, viruses, fungi, mycoplasmas, and mycobacteria. Of these 20 polyps, eight were sterile by all tests, one grew Cryptococcus albidus, one grew Sporobolomyces, one had large numbers of Peptostreptococcus micros and Propionibacterium acnes, greater than 10(6) colony-forming units per gram (cfu/gm), and nine had aerobic bacteria including 10 different species at levels less than 10(5) cfu/gm. The second 20 polyps were cultured for aerobic bacteria only; 11 polyps were positive. Overall, 14 of 26 polyps from patients with asthma and two of 14 polyps from patients without asthma were positive for aerobic bacteria at levels greater than 10(3) cfu/gm (p less than 0.05). Multiple aerobic bacterial species tended to occur in polyps from patients with asthma (11 of 26) more frequently than in those from patients without asthma (one of 14) (p less than 0.01). There was a highly significant positive correlation between tissue neutrophilia and bacterial count (r = +0.9; p less than 0.001). The results indicate that patients with asthma have a significantly higher number and a tendency to a greater variety of aerobic bacteria in nasal polyp tissue than patients without asthma and that the number of infiltrating neutrophils is directly related to the number of bacteria.

Increased numbers of hypodense eosinophils in the blood of patients with bronchial asthma.

We investigated the density of blood eosinophils from patients with asthma using polyvinylpyrrolidone-coated silica gel (Percoll) discontinuous density gradient centrifugation of peripheral blood leukocytes. Ten patients with allergic asthma, 10 normal subjects, and 2 patients with the hypereosinophilic syndrome (HES) were studied. The density distribution profiles of eosinophils from normal subjects showed: (1) peaks at densities of 1.085 to 1.090 g/ml and (2) inflection points or nadirs near 1.082 g/ml, below which only 10% of eosinophils were found. On the basis of these results, we divided eosinophils into 2 subpopulations: normodense (greater than 1.082 g/ml) and hypodense (less than 1.082 g/ml). Densities of eosinophils from patients with asthma and HES peaked at 1.083 and 1.076 g/ml (mean values), respectively, significantly lighter than eosinophils from normal donors (p less than 0.005 and p less than 0.001, respectively). The proportions of hypodense eosinophils in patients with asthma and HES were 35 and 95%, respectively, and were significantly greater than that in normal donors (p less than 0.002 and p less than 0.001, respectively). The density distribution profiles of a normal subject were stable over time, but those of asthmatic patients varied with time. For the 22 participants, there was a positive correlation between log-transformed blood eosinophil counts and the percentage of hypodense eosinophils (r = +0.86, p less than 0.001). Similarly, for 15 of them, plasma eosinophil granule major basic protein correlated with the numbers of peripheral blood eosinophils (r = +0.92, p less than 0.0005) and hypodense eosinophils (r = +0.92, p less than 0.0005). Thus, a portion of the eosinophils in asthmatic patients and most of the eosinophils in patients with HES are hypodense.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium ionophore A23187 calcium-dependent cytolytic degranulation in human eosinophils.

The divalent cation ionophore A23187 is frequently used for studies of eosinophil degranulation. Nonetheless, the mechanism whereby A23187 induces degranulation in human eosinophils is still unclear. In the present experiments, A23187 caused human eosinophils to release a granule protein, eosinophil-derived neurotoxin (EDN) and a membrane-associated protein, Charcot-Leyden crystal (CLC) protein in a calcium and a concentration-dependent manner. However, A23187 at a concentration (1 microgram/ml) that caused 15% EDN release and 30% CLC protein release also produced release of the cytoplasmic enzyme lactic dehydrogenase (LDH) and loss of cell viability, both of which were calcium dependent. CLC protein release preceded EDN release and was detectable even at 15 min after the addition of 1 microgram/ml A23187, whereas EDN release occurred after a lag period of 30 min, and coincided with LDH release. At 1 microgram/ml A23187, neither the release of LDH nor the loss of viability occurred with purified neutrophils obtained in the same blood sample as a by-product of eosinophil purification. Electron microscopic examination demonstrated that exposure to A23187 for 15 min resulted in an increase and elongation of microridges on the cell surface, and exposure for 45 min caused cell disruption followed by extrusion of membrane-bound granules through breaks in the plasma membrane. Only once was granule exocytosis observed. These results indicate that A23187 treatment of eosinophils causes an initial release of membrane-associated CLC protein by a noncytolytic mechanism, and causes degranulation as a result of eosinophil lysis.

Serum IgD in subjects with and without chronic obstructive pulmonary disease: a previous finding restudied.

A previous study of matched pairs of subjects with and without COPD showed higher geometric mean values of serum IgD among the affected subjects. In an attempt to replicate this finding in another sample a cooperative study was undertaken to measure IgD in subjects from an epidemiologic study of airway obstructive disease from a different geographic region. The relationship between serum IgD (Ine scale) and % FEV1, was identical in the 2 samples. The lack of statistical significance when analyzing the Arizona sample in a matched pair fashion is attributed to an underrepresentation of subjects with very low % FEV1, and smaller sample size.

Quantification of serum IgE in patients with burns.

IgE levels in the serum of individuals with burns were sequentially measured and compared to IgE levels in normal control blood donors. Following a burn, the levels of IgE protein showed a significant, although modest, increase, usually evident of IgE protein showed a significant, although modest, increase, usually evident between days 14 and 22. In an occasional patient, the IgE levels rose by as much as five times in value during this period. Because of the complexity of the clinical situation associated with the burn patients, we cannot ascribe these elevations of IgE to the burn per se, but must consider the possibility of other factors, especially those involved in the treatment of the burn as well as the infection. The magnitude of the elevation of IgE in the burn patients (geometric mean=272 ng/ml) was considerably lower than the magnitude of the elevations seen in atopic dermatitis and generalized neurodermatitis (geometric means=2265 and 2071 ng/ml, respectively). Thus simple trauma to the skin is not a sufficient explanation for the elevated serum IgE levels in atopic dermatitis and generalized neurodermatitis.

Immunoglobulin concentrations in serum and nasal secretions in chronic obstructive pulmonary disease. A matched-pair study.

Immunoglobulin concentrations were determined in the sera and nasal washes of 111 patients with chronic obstructive pulmonary disease who were 45 to 60 years of age and in 111 control subjects matched with the patients for age, sex, occupation, and smoking history who demonstrated normal 1-sec forced expiratory volume. Serum IgA, IgM, IgG, nad IgE were not significantly different in the 2 groups. Serum IgD was significantly higher in subjects with chronic obstructive pulmonary disease. Nasal wash IgD and IgM, expressed as percentages of total protein, were higher in index cases, but nasal wash IgA and IgG were comparable in both groups. The finding of relatively high concentrations of IgA, expressed as fractions of total protein, in respiratory secretions compared to serum is consistent with earlier findings that IgA is actively secreted from the respiratory epithelium and is not deficient locally in subjects with chronic obstructive pulmonary disease. In contrast, IgM and IgG expressed as proportions of total protein were consistently higher in sera than secretions. The IgE in nasal secretions was detected so seldom in this study that too few matched pairs were available for statistical analysis. The higher concentration of IgD in the serum and nasal secretions of patients with chronic obstructive pulmonary disease compared with their matched pairs and the associated higher frequency of low IgD in control subjects suggests that low IgD may be protective against the development of chronic obstructive pulmonary disease. Further studies on the biologic role of IgD may provide better understanding of these findings.

Inheritance of low serum immunoglobulin D.

Previous studies have shown that log IgD levels in normal individuals are distributed in a nonunimodal manner. Therefore, in this study we tested whether inheritance might play a role in determination of IgD levels. IgD levels were measured in serum or plasma from 301 randomly selected children aged 6-18 yr, 245 consecutive adult blood donors, and 134 first-degree relatives of subjects with low IgD levels. Comparison of serum and plasma from five individuals revealed no difference, so the two were used interchangeably. The distributions of log IgD levels in randomly selected populations of both adults and children were nonunimodal with nadirs at 2.15 IU/ml. In both of these randomly selected populations, 13-14% of the subjects had low IgD values (<2.15 IU/ml). In addition, there was a significant sibling-sibling correlation of log IgD values (r = 0.56, n = 72, P <0.01). Because of the nonunimodality of the frequency distribution histogram for IgD values and because of the familial aggregation of these values, the study was extended to include first-degree relatives of subjects with low plasma IgD. Blood samples from 92% of living first-degree relatives, 134 individuals, were analyzed for their level of IgD, and the results of segregation and pedigree analyses of these data were compatible with autosomal recessive inheritance of an allele for low plasma IgD levels. IgD values in plasma from siblings of probands for low IgD were also non-unimodal in distribution with a nadir at congruent with2.15 IU/ml. The results suggest that there is autosomal recessive inheritance of an allele for low plasma IgD.

Measurement of IgD by a double antibody radioimmunoassay: demonstration of an apparent trimodal distribution of IgD levels in normal human sera.

A double antibody radioimmunoassay (RIA) was developed to measure IgD in serum and secretions. One IgD myeloma protein was used as radiolabeled antigen and standard with antiserum to a second IgD myeloma protein. The IgD standard and normal sera yielded parallel inhibition curves in the RIA and inhibition was produced by IgD and not by any of the other immunoglobulins. The assay had a lower limit of sensitivity of 0.01 International Unit (I.U.)/ml and modifications increased the sensitivity to 0.0008 I.U./ml. Measurable IgD levels were found in all 112 normal adult sera assayed (geometric mean 13.0 I.U./ml, arithmetic mean 30.1 I.U./ml, median 14.8 I.U./ml, range 0.10 to 202 I.U./ml, 95% of values between 0.19 and 156 I.U./ml. The distribution of IgD in the 112 normal sera appeared trimodal with modes at approximately 0.25 I.U./ml, 5I.U./ml, and 35 I.U./ml. IgD was measurable in nasal and bronchial washes and human milk, but could not be detected in parotid fluid.

Comparison of procedures for measurement of IgE protein in serum and secretions.

Because of conflicting reports in the literature concerning the value of various procedures for measurement of IgE in serum and secretions, we compared four different methods, the radioimmunosorbent test (RIST), the double-antibody radioimmunoassay (RIA), the paper disc immunosorbent test (PRIST), and radial immunodiffusion (RID). The standards used in the assays were tested initially in the double-antibody RIA with the use of the reference from the World Health Organization. The results showed that RID as expected was relatively insensitive and IgE was reliably measured only above approximately 1,000 international units (IU). Moreover, certain sera containing low levels of IgE by the other procedures gave distinct precipitin zones and presumably falsely high levels of IgE protein. Thus RID may yield apparently erroneous results when used as a screening procedure for measurement of IgE levels. Among the other procedures PRIST and the double-antibody RIA showed the best agreement. With serum samples RIST yielded values for IgE in the low level range higher than those given by the PRIST and double-antibody RIA. With breast milk and colostrum, values of IgE between 120 and 690 ng/ml were found by RIST, whereas IgE was not detected by double-antibody RIA and PRIST. No evidence of an inhibitor of IgE was found in breast milk, so that the apparent elevation of IgE in breast milk by the RIST is likely false. These findings confirm prior reports of spurious elevations of IgE with the RIST and indicate the usefulness of the PRIST and double-antibody RIA for the measurement of IgE in sera and secretions.