Cutaneous basophil (Jones-Mote) hypersensitivity after "tolerogenic" doses of intravenous ovalbumin in the guinea pig.

Cutaneous basophil hypersensitivity (CBH) was studied for tolerogenic requirements. Graded doses of intravenous ovalbumin (OA) were given to guinea pigs which were subsequently immunized appropriately to produce CBH, classic delayed hypersensitivity (classic DH), and/or antibodies of both passive cutaneous anaphylaxis (PCA) and hemolytic types. Results showed that doses of intravenous antigen sufficient to induce subsequent tolerance for classic DH and hemolytic antibody actually stimulate CBH reactivity and PCA antibody production. Other studies of dose-route relationships for CBH production demonstrated that optimal immunogenic dosage requirements for CBH varied widely with route of antigen employed. OA in incomplete Freund's adjuvant (IFA) injected into footpads had low dosage requirements, intravenous OA had high dose requirements, and intradermal soluble OA dosage requirements were intermediate. The observation that blatant immunogenic responses occur during the early period of tolerance induction amplifies the significant heterogeneity of the cellular immune response and may be of importance in understanding tolerogenesis. Similar immunogenic-tolerogenic requirements and the prime role played by the basophil suggest a developmental or functional relationship between CBH and PCA antibody response.

Cutaneous basophil hypersensitivity. I. A new look at the Jones-Mote reaction, general characteristics.

Jones-Mote reactivity, defined as a delayed-type skin reaction, occurs transiently early in the course of immunization with protein antigens or hapten conjugates with or without the adjuvant effect of tubercle bacilli. The skin reaction is typically a flat, well-circumscribed erythema with little induration beginning at about 6 hr, reaching a peak at 18-24 hr, and fading or gone at 48 hr. Immunogenic carrier requirements for hapten-specific Jones-Mote hypersitivity resemble those of antibody production rather than of classic delayed hypersensitivity. Skin test antigen requirements indicate that the Jones-Mote reaction involves an active stimulatory response rather than combination with preformed antibody, since ABA conjugates of nonimmunogenic D-polymers do not work. Studies with ALS and carrageenan suggest that the lymphocyte is an important contributor to the reaction, but the macrophage is not. Because the reactions studied here are operationally different from those described by Jones and Mote and because they have a characteristic histology, the term "cutaneous basophil hypersensitivity" is proposed.

Cutaneous basophil hypersensitivity. II. A light and electron microscopic description.

Delayed onset erythematous skin reactions elicited in guinea pigs early in the course of sensitization with azobenzenearsonate-protein conjugates or with protein antigens in incomplete Freund's adjuvant or in saline were found to have a characteristic morphology which sets them apart from delayed hypersensitivity and the classic antibody mediated reactions. The principle feature was massive dermal infiltration with basophilic leukocytes. Mononuclear cells of several types including activated and small lymphocytes, monocytes, macrophages, and blast cells were also present. Such reactions have in the past been designated Jones-Mote hypersensitivity, but we prefer the descriptive term cutaneous basophil hypersensitivity (CBH) for the reasons given. Occasional basophils extruded their granules, and individual granules, retaining their characteristic ultrastructure, were commonly seen in the interstitium. However, intercellular junctions between endothelial cells were closed except during cell emigration and there was no morphologic evidence of an histamine-like effect. The majority of basophils, moreover, did not degranulate but underwent nuclear pyknosis and cytoplasmic degeneration and were phagocytosed by macrophages. Phagocytosed basophil granules retained their ultrastructure. Skin tests performed at late intervals after sensitization had a different time course and morphology. Animals sensitized with protein antigens in complete Freund's adjuvant developed delayed hypersensitivity; however, reactions elicited in such animals at early (but not late) intervals after sensitization contained a prominent basophil component. We interpret such reactions to be a mixture of delayed hypersensitivity and cutaneous basophil hypersensitivity. The function of the basophil in CBH and its relation to the mononuclear cells which accompany it are unknown, and various possibilities are discussed. We conclude that cutaneous basophil hypersensitivity is a distinct immunologic and morphologic entity, occurring early in the course of sensitization with protein antigens incorporated in any of several vehicles. The mechanism of the reaction is presently unknown, and a general hypothesis to explain its pathogenesis has been proposed.

Direct evidence of a role for mast cells in the pathogenesis of antigen-induced bronchoconstriction.

We measured bronchoalveolar lavage (BAL) fluid histamine levels in allergic asthmatics and nonallergic normal subjects after local airway antigen and cold 22 degrees C normal saline challenges. Immediately after instillation of antigen through a bronchoscope wedged into a subsegmental airway, all 17 allergic asthmatics but none of the nine normal subjects had visible airway constriction. The asthmatics had a concomitant mean increase in BAL histamine of 23% (P = 0.005), whereas the normals had no change in BAL histamine. Among the allergic asthmatics, the change in BAL histamine content in response to antigen directly correlated with the control (baseline) BAL histamine content (r = 0.66, P = 0.003). Moreover, asthmatics with large antigen-induced changes in BAL histamine had greater airway methacholine sensitivity than did asthmatics without measurable increases in BAL histamine (8 +/- 2 vs. 41 +/- 31 breath units). Neither asthmatics nor normal subjects had airway constriction or changes in BAL histamine levels in response to nonspecific challenge with cold saline. Our data suggest that when allergic asthmatics are exposed to relevant antigens they have in vivo lung mast cell degranulation which results in airway constriction and contributes to nonspecific airway hyperresponsiveness.

Elevated bronchoalveolar lavage fluid histamine levels in allergic asthmatics are associated with methacholine bronchial hyperresponsiveness.

Using a sensitive single isotope enzymatic assay we measured bronchoalveolar lavage (BAL) fluid histamine in asymptomatic normal (nonallergic), allergic rhinitic, and allergic asthmatic subjects. Normal subjects were found to have little or no detectable amounts of histamine in BAL fluid (11 +/- 11 pg/ml), and few BAL fluid mast cells. In comparison, the allergic rhinitics and allergic asthmatics had much higher amounts of BAL fluid histamine (113 +/- 53 and 188 +/- 42 pg/ml, respectively), and a significantly greater number of BAL fluid mast cells. Furthermore, despite having equivalent baseline pulmonary function values, allergic asthmatics with BAL fluid histamine levels greater than 100 pg/ml required only 7 +/- 2 breath units of methacholine to induce a 20% drop in forced expiratory volume in 1 s (FEV1) (PD20FEV1) while asthmatics with BAL fluid histamine levels less than 100 pg/ml required 49 +/- 19 breath units (P less than 0.05). These data suggest that allergic asthmatics have ongoing lung mast cell degranulation that might contribute to the etiology of airway hyperresponsiveness.

Oncofetal protein accompanying irradiation-induced small-bowel adenocarcinoma in the rat.

A tumor-associated protein was found in tissue derived from an X-irradiation-induced adenocarcinoma in the small bowel of the rat. The protein was associated with the cell membranes of the tumor tissue. It shared common antigenic determinants both with a rat fetal protein and a perchloric acid-soluble protein isolated from the serum of the tumor-bearing rat.

The antigen-limited nature of microtiter ELISAs requires partial depletion of IgG to permit reliable determination of rabbit serum IgA antibody activity.

The antigen-limiting nature of microtiter ELISAs predicts that antibodies of minor classes may be underestimated when the same specimen contains large amounts of IgG antibodies specific for the same antigen. Such competitive inhibition can be diagnosed from ELISA titration plots. A method is described to eliminate the negative effects of this competition on the detection of IgA antibodies in rabbit serum. The detectability of rabbit serum antibodies to ovalbumin and bovine serum albumin is increased 10-fold by prior treatment of 1:100 dilutions of serum with 1% Cowan I S. aureus. High concns of S. aureus, e.g. 10%, completely deplete serum IgG without loss of IgA. However, concns higher than 1% do not lead to additional improvement in the detectability of IgA antibodies in the systems studied. The method is rapid, inexpensive and shows no non-specific depletion of IgA from either serum or bronchoalveolar lavage fluid.

Altered recognition of surface-adsorbed compared to antigen-bound antibodies in the ELISA.

We found in preliminary studies using 125I-labelled antibodies that an antibody bound to a solid-phase antigen was recognized more efficiently than an antibody adsorbed directly to the solid phase. The present study was designed therefore to quantitate the differential recognition of an antibody adsorbed directly to the solid phase and an antibody bound to antigen on the solid phase using the amplified enzyme-linked immunosorbent assay (a-ELISA), and to compare results with the amounts of specific antibody determined by quantitative immunoprecipitation. The degree of differential recognition was quantitated for rabbit IgG and SIgA anti-ovalbumin (anti-OA) and anti-fluorescein, and was found to be dependent upon the isotype of the antibody and not its specificity. The ratio describing the differential recognition of SIgA antibodies (1.8) was much less than for IgG antibodies (greater than 30) and remained constant over the titration range analyzed while the ratios obtained for IgG varied substantially (25-60) over the same range. These ratios of differential recognition were used to estimate rabbit IgG antibody levels to OA, bovine serum albumin, ferritin and alpha-lactalbumin. The estimates obtained were consistently much less than total antibody levels measured by quantitative precipitation. The use of glutaraldehyde-aggregated OA in the ELISA, however, increased the amount of IgG anti-OA and SIgA anti-OA capable of recognizing OA adsorbed on plastic from 12 to 50 and from 30 to 80%, respectively.

Synthesis of fluorescent muramyl dipeptide congeners. 2.

Muramyl dipeptide (MDP) analogues were prepared and utilized in the synthesis of new fluorescently labeled MDP derivatives for use as biologic probes. Thus, N alpha-(N-acetylmuramyl)-L-lysyl-D-isoglutamine (Lys-MDP, 4) and N alpha-(N-acetylmuramyl)-L-alanyl-D-isoglutaminyl)-L-lysine [MTP, 5] were synthesized and then reacted with 2-(fluoresceinylamino)-4,6-dichloro-s-triazine (DTAF, 2) to yield the fluorescent adducts, DTAF-Lys-MDP (6) and DTAF-MTP (7). The adjuvant activity of the fluorescent MDP derivatives was determined by the ability of the compounds to promote delayed skin test responses in guinea pigs immunized with ovalbumin (OA) and by evaluating the anti-OA activity of these guinea pigs.

Synthesis of a biologically active fluorescent muramyl dipeptide congener.

A fluorescent-labeled muramyl dipeptide (MDP) has been prepared to probe immunoadjuvant cellular interactions. N-Acetylmuramyl-L-alanyl-D-isoglutamine (1) was synthesized in improved yield and reacted with 2-(fluoresceinylamino)-4,6-dichloro-s-triazine (DTAF, 2) to give the fluorescent adduct DTAF-MDP (3), attached through the 6-position of the sugar moiety. Adjuvant activity was assessed by using two different in vitro assays, macrophage spreading, and inhibition of macrophage migration. Both assays indicated that the apparent adjuvant activity of 3 is comparable to that of 1.

Site of airflow obstruction during early and late phase asthmatic responses to allergen bronchoprovocation.

Chronic asthma and late asthmatic responses (LAR) are associated with local inflammation which might be expected to produce airflow obstruction in small airways and to increase nonspecific airway reactivity. In contrast, early asthmatic responses (EAR) are primarily bronchospastic and probably involve more central airways. We challenged 17 nonsmoking, mildly asthmatic atopic subjects with allergen bronchoprovocation and measured changes in spirometry (FEV1) over the next 24 hours. Each subject also performed a helium-oxygen (He-O2) flow-volume loop before challenge (baseline), during the EAR, and six hours and 24 hours after challenge to measure the effect of gas density on flow rates at midvital capacity. Twelve subjects had both an EAR and a LAR; five subjects had only a LAR. Of these 17 subjects, 15 were initially density dependent, while only two were density independent. During the EAR, 13 percent of the density dependent population had significant decreases in delta Vmax 50 percent; 47 percent had significant decreases during LAR. The He-O2 flow data analyzed at specified time points after challenge revealed significant decreases in the mean delta Vmax 50 percent at six hours in those who had only a LAR (p less than 0.01). In those who had a dual airway response, density dependence increased during the EAR, but decreased at six and significantly at 24 hours (p less than 0.05) postchallenge. There was a strong trend for the severity of the LAR (measured by changes in FEV1) to be directly related to the total decrease in delta Vmax 50 percent during the LAR. We conclude that late asthmatic responses occur frequently after a single antigenic bronchial challenge and can be associated with persistent symptoms. The LAR were often associated with a decrease in density dependence of maximal expiratory airflow, and therefore, may involve small airways.

Experimental leukocyte-induced pulmonary vasculitis with inquiry into mechanism.

Intravenous transfusions of washed allogeneic or autologous leukocytes in rabbits resulted in lesions of pulmonary periarteritis 48 hours later. Intact leukocytes were required. Systemic anaphylaxis, generalized Shwartzman reaction, alternate pathway complement activation and inert particle microembolism failed to produce identical lesions. Leukocytes tagged with radioactive chromium were found within arterial thromboses with proximal vasculitis. Generation or release of inflammatory factors plus thromboembolism would explain the pathogenesis of the lesions described. Specific mechanisms may be quite complex. Similar lesions have not been described in studies of pulmonary leukocyte entrapment or experimental microembolism of the lung. This model may be useful for studying pathogenetic mechanisms in pulmonary vasculitis and may have clinical implications.

Bronchoalveolar lavage of allergic asthmatic patients following allergen bronchoprovocation.

In these studies, we describe the use of bronchoalveolar lavage (BAL) to study local changes following aerosol bronchoprovocation (BPC) and environmental exposure to antigen in mildly symptomatic asthmatic patients. The BAL was performed in 12 atopic subjects "out of season," and in five normal subjects at baseline, less than or equal to 4, or 24 hours following BPC. Five asthmatic patients were also lavaged during seasonal exposure to allergen. The BAL cells were examined with light and transmission electron microscopy. Bronchoprovocation, by itself, resulted in an average maximal decrease in FEV1 of 13 percent just prior to BAL. There was no significant decrease in FEV1 as a result of BAL. Within four hours after BPC, the number of neutrophils was significantly greater in BAL compared to baseline (1.5 +/- 0.6 X 10(5) vs 3.4 +/- 1.7 X 10(5) cells; p less than 0.01), and the number of eosinophils was significantly greater within four hours and at 24 hours when compared to baseline values (0.4 +/- 0.3 X 10(5) vs 1.9 +/- 0.7 X 10(5) vs 1.2 +/- 0.4 X 10(5) cells; p less than 0.02). Transmission electron micrographs of BAL from lungs of asthmatic patients revealed degranulation of mast cells and loss of core material from eosinophil granules following challenge with aerosolized allergen or with spontaneous environmental exposure. These studies show that in carefully selected, mildly symptomatic asthmatic subjects, BPC and BAL may be useful to evaluate pathogenetic mechanisms in allergic bronchial asthma.

Use of segmental airway lavage to obtain relevant mediators from the lungs of asthmatic and control subjects.

Studies have demonstrated that increased amounts of histamine in the airways of asthmatic patients are associated with increased airway reactivity. However, using routine bronchoalveolar lavage (BAL), histamine can be detected in only a portion of asthmatic subjects and a minority of control populations. To obtain relevant mediators from the airways in higher concentrations by avoiding the dilution inherent with a standard BAL, a technique was developed to lavage isolated airway segments of the human lung that employed a double-lumen bronchoscope and a balloon-tipped catheter. Lavage fluid obtained by this method yielded significantly higher concentrations of histamine than that obtained with routine BAL (asthmatic subjects, 2,403 +/- 633 pg/ml vs 188 +/- 42 pg/ml; rhinitis subjects, 533 +/- 187 pg/ml vs 113 +/- 53 pg/ml; normal subjects, 174 +/- 63 pg/ml vs 11 +/- 11 pg/ml). Similar findings were also noted for prostaglandin D2 (PGD2). Segmental airway lavage also resulted in higher lavage fluid concentrations of LTB, than routine BAL. Segmental airway lavage should help in studying the relationship of mast cell degranulation to airways reactivity in both asthmatic and other study populations.

Methods for bronchoalveolar lavage in asthmatic patients following bronchoprovocation and local antigen challenge.

We employed bronchoalveolar lavage (BAL) of subsegmental airways to study the local inflammatory effects of aeroallergen bronchoprovocation (BPC) and local instillation of allergen in allergic asthmatic patients, allergic rhinitis patients, and normal subjects. Two protocols were used: (1) BAL was performed in three subsegments following BPC or during spontaneous seasonal exposure, and (2) 5-ml aliquots of increasing doses of allergen were instilled into a single subsegment until there was at least 30 percent closure of the airway; the airway was then immediately lavaged. A subsegment in the opposite lung was lavaged as a control site. These same two segments were lavaged again two to 14 days later and the cells and fluid analyzed. Fifty-five lavages have been performed without complications. Pulmonary function tests (FEV1) were not significantly disturbed by either local challenge or lavage procedures. Cells were examined using light and electron microscopy and showed inflammatory cells in alveolar airways and dissolution of mast cell and eosinophil granules. Using selected criteria, we were able to use these methods in mildly, seasonally asthmatic patients to obtain safely cells and fluid for analysis. These techniques may permit studies which further our understanding of the mechanisms responsible for allergic asthma.

Protamine-induced fatal anaphylaxis. Prevalence of antiprotamine immunoglobulin E antibody.

Protamine is used widely to reverse the anticoagulant effects of heparin and to delay the absorption of insulin. Although adverse reactions to protamine are reported infrequently and are usually mild, we recently observed the first fatal case of type I anaphylaxis resulting from protamine. This patient had previously been sensitized to protamine during cardiac catheterization and had high levels of protamine-specific immunoglobulin E in the serum. In a prospective study, we found that 10 of 19 diabetic patients (53%) who had received insulin containing insulin also had high levels of antiprotamine immunoglobulin E. In contrast, none of 27 nondiabetic healthy normal controls or 10 diabetics who had never received protamine or protamine-containing insulin had levels of antiprotamine immunoglobulin E over background. This study underscores the risks of routinely administering protamine to susceptible individuals and the need for alternative therapies.

Increased bronchovascular permeability after allergen exposure in sensitive asthmatics.

To investigate the possibility that an increase in bronchovascular permeability is associated with allergen exposure in sensitive asthmatics we evaluated the amounts of serum proteins in bronchoalveolar lavage (BAL) effluents before and after local challenge with allergen. After exposure of sensitive asthmatic airways (n = 15) to allergen significant increases in total protein compared with controls were observed: 0.08 +/- 0.01 mg/ml in control airways and 0.13 +/- 0.02 mg/ml in challenged airways; P less than 0.05. The greatest changes induced by allergen exposure involved small-molecular-weight proteins (less than 345,000) and an inverse correlation was observed between log molecular weight and percent increase in the concentrations of the specific proteins; r = -0.61. BAL-serum distribution coefficients of serum proteins in airway fluids reflected a greater diffusability of low-molecular-weight proteins immediately after allergen exposure. We also evaluated the movement of serum proteins into lung after local allergen exposure using intravenously administered 99mTc-albumin (n = 10) and found an immediate 3.8-fold increase in amounts of radioactive albumin in allergen exposed airways compared with airways exposed to diluent. Most of the radioactivity was recovered in the first 5 ml of aliquot withdrawn, suggesting a marked increase in the permeability of the bronchial (large airway) vascular-epithelial membrane. An increase in serum proteins was also observed in BAL fluid of asthmatics 2-4 h after aerosol challenge (n = 4), including all proteins in the molecular weight range 45,000-900,000. These studies suggest that allergen exposure in sensitive asthmatics causes an acute increase in bronchovascular permeability to serum proteins.

Allergic alveolar diseases. Problems in diagnosis and management.

One of the diagnostic possibilities to consider when a patient presents with cough, fever, dyspnea, or pulmonary infiltrates is hypersensitivity pneumonitis. Some of the problems encountered in diagnosis of diffuse lung disease are illustrated in two case reports. In one of the cases, interstitial pneumonitis of insidious onset was attributed to inhalation of thermophilic organisms in moldy silage. In the other, the outstanding pathologic feature was bronchiolitis obliterans, and circumstantial evidence pointed to a home humidifier as the source of the problem.

Immunology of Allergy.

Immune reactions, presumably developed to rid organisms of troublesome invaders, are rather frequently associated with responses that result in injury to host tissue. Such responses are manifestations of allergy or hypersensitivity, and involve antibodies of certain immunoglobulin classes, complement components, mast cells and basophils, lymphocytes, macrophages, and various pharmacologic mediators and other soluble substances in an exuberant array of possible combinations. An understanding of clinical hypersensitivity diseases is aided by classifying basic allergic mechanisms into four main types: anaphylactic (Type I), cytotoxic (Type II), complex-mediated (Type III), and cell-mediated (Type IV), which may participate in various combinations in disease states.