Burton Zweiman

The alpha form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis.

Tryptase, a protease produced by all mast cells, was evaluated as a clinical marker of systemic mastocytosis. Two sandwich immunoassays were evaluated, one which used the mAb G5 for capture, the other which used B12 for capture. The B12 capture assay measured both recombinant alpha- and beta-tryptase, whereas the G5 capture assay measured primarily recombinant beta-tryptase. G5 binds with low affinity to both recombinant alpha-tryptase and tryptase in blood from normal and nonacute mastocytosis subjects, and binds with high affinity to recombinant beta-tryptase, tryptase in serum during anaphylaxis, and tryptase stored in mast cell secretory granules. B12 recognizes all of these forms of tryptase with high affinity. As reported previously, during systemic anaphylaxis in patients without known mastocytosis, the ratio of B12- to G5-measured tryptase was always < 5 and approached unity (Schwartz L.B., T.R. Bradford, C. Rouse, A.-M. Irani, G. Rasp, J.K. Van der Zwan and P.-W.G. Van der Linden, J. Clin. Immunol. 14:190-204). In this report, most mastocytosis patients with systemic disease have B12-measured tryptase levels that are elevated (> 20 ng/ml) and are at least 10-fold greater than the corresponding G5-measured tryptase level. Most of those subjects with B12-measured tryptase levels of < 20 ng/ml had only cutaneous manifestations. The B12 assay for alpha-tryptase and beta-tryptase, particularly when performed in conjunction with the G5 assay for beta-tryptase, provides a more precise measure of mast cell involvement than currently available assessments, a promising potential screening test for systemic mastocytosis and may provide an improved means to follow disease progression and response to therapy.

Isolation of myelin basic protein-reactive T-cell lines from normal human blood

T-Cell lines which responded by proliferation to the autoantigen, myelin basic protein (MBP), were isolated from the blood of six of nine normal humans. These T-cell lines could be maintained in in vitro culture for up to 2 months through the use of Interleukin 2 and repeated MBP stimulation. Optimal antigen-induced proliferation required both antigen and antigen-presenting cells found in the adherent cell population of autologous peripheral blood mononuclear cells (PBM). The T-cell lines were predominantly of the helper phenotype (OKT3+, OKT4+, OKT8-) and responded to both human and guinea pig myelin basic protein.

Release of Neutrophil Chemotactic Activity during Immediate Hypersensitivity Reactions in Humans

Heat-stable, serum-derived chemotactic activity for neutrophils is shown in a human model of immunoglobulin E-mediated asthma. Twenty-six ragweed-sensitive subjects underwent bronchial provocation challenge using ragweed and Mecholyl. Increased neutrophil chemotactic activity was found in serum tested from 5 to 30 min after a positive ragweed-inhalation challenge, but not after negative ragweed challenge. The appearance of neutrophil chemotactic activity did not reflect the effects of bronchospasm alone, because it was not found after bronchospastic responses to Mecholyl in the same subjects. There were no accompanying changes of serum complement activity, nor evidence of inhibition of the chemotactic activity by proir exposure to antisera to the third and fifth components of complement. Ultrafiltration of serum showed chemotactic activity contained in fractions of at least 50 000 daltons. This appears to be the first demonstration of neutrophil chemotactic activity liberated during experimentally induced immunoglobulin E-mediated asthma in humans.

In Vitro Cell-Mediated Immunity of Cerebrospinal-Fluid Lymphocytes to Myelin Basic Protein in Primary Demyelinating Diseases

In an attempt to characterize the immunologic reactivity of cerebrospinal-fluid lymphocytes in demyelinating diseases, we compared the myelin-basic-protein-induced in vitro responses of these cells to peripheral blood lymphocytes from the same subjects with a variety of neurologic diseases. Peripheral blood lymphocytes from patients with acute disseminated encephalomyelitis and progressive multiple sclerosis had increased reactivity as compared to those of normal volunteers (P less than 0.01 and P less than 0.05, respectively). Cerebrospinal-fluid lymphocytes from patients with acute disseminated encephalomyelitis and acute and progressive (but not stable) multiple sclerosis were more reactive than cells from subjects with other neurologic diseases (P less than 0.005, P less than 0.02 and P less than 0.05, respectively). Cerebrospinal-fluid lymphocytes manifested a greater reactivity than peripheral blood lymphocytes in acute and progressive multiple sclerosis but not in acute disseminated encephalomyelitis. These findings demonstrate that lymphocytic cells reactive to myelin basic protein are present in the spinal fluid during active demyelinating disease; and that these cells may be more reactive than peripheral blood lymphocytes.

The thymus in myasthenia gravis. Evidence for altered cell populations

Abstract Comparisons of several types of immunologic reactivity were made in thymic cells from six patients with myasthenia gravis and thymic hyperplasia, four patients with myasthenia gravis and thymoma and six age-matched control cardiac-surgery patients. In mixed leukocyte reactions, thymic cells from the subjects with hyperplasia were capable of stimulating autologous peripheral blood lymphocytes. Such reactivity was not seen in thymocyte-blood lymphocyte mixtures from the other two groups. There was an increased number of B cells in the thymic-cell populations from the myasthenic patients as compared to that in the control group, carrying predominantly IgM receptors. The thymic cells from myasthenic patients also responded more vigorously to pokeweed mitogen. These findings suggest altered populations of lymphoid cells in the thymus of myasthenic patients that react with autologous lymphocytes from other cell compartments. The pathogenic implications of these findings remain to be determined. (N Engl...

Histamine suppression of human lymphocyte responses to mitogens.

Abstract Exogenously added histamine in non-cytotoxic concentrations (10−5−10−3M) suppresses in vitro proliferation of lymphocytes induced by PHA or Concanavalin A. This suppressive effect was observed when histamine was present for as short as 1 2 hr in the beginning of the culture. Histamine, in concentrations as high as 10−3M, did not cause increased release of isotope from 51Cr-labeled lymphocytes following 4 hr of incubation. The histamine H2 receptor antagonist, metiamide, but not the H1 receptor antagonists diphenhydramine or chlorpheniramine, blocked the histamine suppressive effect. Some of the biological implications of these findings are discussed.

Antigen-induced neutrophil chemotactic activity in man: Correlation with bronchospasm and inhibition by disodium cromoglycate

We have previously reported increased neutrophil chemotactic activity in sera obtained after positive antigen inhalation responses in atopic subjects. This report describes the kinetics of appearance of this serum activity and the effects of antigen dose and disodium cromoglycate pretreatment on the response in 10 ragweed-sensitive subjects. Significantly increased chemotactic activity was present as early as 1 min, peaked at 10 min, and persisted through 24 hr after inhalation of antigen. The increased chemotactic activity correlated with the degree of bronchospasm induced by antigen inhalation and the amount of antigen administered. The increased chemotactic activity and bronchospasm were blocked by administration of disodium cromoglycate prior to antigen challenge. These findings are consistent with a postulated antigen-induced anaphylactic release of chemotactic activity. The correlation of this activity with the degree of bronchospasm and its appearance after administration of even small doses of antigen suggest that this activity may be important in antigen-mediated bronchospasm.

Accumulation of Leukotriene C4 and Histamine in Human Allergic Skin Reactions

To determine whether lipoxygenase products of arachidonic acid metabolism are released in vivo during human allergic cutaneous reactions, we serially assayed chamber fluid placed over denuded skin sites for the presence of both C-6 peptide leukotrienes (e.g., LTC4, LTD4, and LTE4) and leukotriene B4 (LTB4), using radioimmune assay and HPLC separation, and compared it to histamine (assayed radioenzymatically) in 13 atopic and two nonatopic volunteers. Skin chamber sites challenged with ragweed or grass pollen antigen (250-750 protein nitrogen units/ml) for the first hour and phosphate-buffered saline (PBS) for the next 3 h were assayed hourly and compared to sites challenged with PBS alone. As assessed by HPLC, LTC4 composed greater than 85% of the C-6 peptide leukotriene released at any skin site, whereas little LTD4 or LTE4 was detected. LTC4 was present in significantly greater concentrations at antigen sites as compared to PBS-challenged sites throughout the 4-h period. Minimal concentrations of LTB4 were found throughout this time period and were not different at antigen or PBS sites. Histamine was present in significantly greater concentrations at antigen rather than PBS sites, but the pattern of release was different from that of LTC4. Peak histamine release invariably occurred during the first hour and decreased progressively thereafter, whereas the greatest amounts of LTC4 were detected during the 2nd to 4th hours. The amount of LTC4 accumulating at the site was dependent upon the dosage of antigen used in the epicutaneous challenge. We have demonstrated in this study that of the leukotrienes assessed LTC4 is released in the greatest quantity in situ during in vivo allergic cutaneous reactions and that it is present at such sites for at least 4 h after antigen challenge. Since intradermal injection of LTC4 in humans induces wheal and flare responses that persist for hours, our findings support the hypothesis that LTC4 is an important mediator of human allergic skin reactions.

Immunohistochemical detection of human basophils in late-phase skin reactions

BACKGROUND Human basophils are difficult to detect with classic histochemical stains at sites of allergic inflammation. The 2D7 anti-basophil monoclonal antibody was used to identify basophils in skin during the late-phase response to a cutaneous allergen challenge. METHODS The 2D7 monoclonal antibody was used on protease-digested sections of skin biopsy specimens obtained 6 and 24 hours after an allergen or buffer challenge. The skin chamber technique was used to compare buffer- and allergen-challenged sites at 6 hours, and intradermal injection of allergen was used to compare allergen-challenged sites at 6 and 24 hours. RESULTS Dramatic increases in the numbers of 2D7+ cells and in tissue staining by 2D7 were observed 6 hours after allergen challenge compared with buffer challenge. Histamine levels in skin chamber fluid varied with 2D7+ cell concentrations. By 24 hours, 2D7+ cells and tissue staining appeared to diminish but were still detectable in the allergen-challenged sites. Basophils localized primarily in and around blood vessels, whereas mast cells remained mostly in the superficial dermis. Mast cells were 2D7- in both the allergen- and buffer-challenged skin. Metachromatic staining of 2D7+ basophils with toluidine blue was absent in these tissue sections. CONCLUSIONS The 2D7 monoclonal antibody provides a more sensitive and precise marker than histochemical staining for human basophil involvement during the late-phase response to an allergen challenge. Basophil infiltration was observed at 6 hours only after allergen challenge and persisted at similar levels by 24 hours.

Histologic studies of human skin test responses to ragweed, compound 48 80 , and histamine

Abstract The patterns of mast cell and eosinophil changes at the site of intradermal injection of ragweed, compound 4880, and histamine were studied by histologic techniques. Dermal biopsies of 23 ragweed skin test-positive subjects revealed increasing numbers of eosinophils and decreasing numbers of mast cells over a 240 minute period following injection of antigen. The eosinophil response began in the periappendigeal areas, extended into the interstitium, and occurred in sites with depressed wheal reactions in hydroxyzine-treated subjects. These cellular patterns did not occur at ragweed skin test sites in nonatopic subjects. However, compound 4880 induced similar eosinophilic responses in both atopics and nonatopics. Histamine injection was followed by no eosinophilic or mast cell changes. These findings support the hypothesis that eosinophil responses in reagin-mediated reactions may occur secondary to release of a mediator other than histamine of mast cell origin.