Mechanisms in the suppression of delayed hypersensitivity in the guinea pig by 6-mercaptopurine.

The mechanism of suppression, of delayed hypersensitivity to tuberculoprotein by 6-mercaptopurine (6-MP) was studied in guinea pigs. Under the conditions of the protocol, suppression of tuberculin delayed skin test reactivity was not associated with a significantly altered end-organ response to mediators of permeability. No significant alteration of in vivo lymphoid activity, as measured by reconstitution studies, was found. In addition, lymphoid cells from 6-MP-treated animals reacted in a fashion similar to those of placebo-treated animals with respect to (a) antigen-induced lymphocyte proliferation, (b) antigen-induced liberation of macrophage inhibitory factor activity, (c) direct inhibition by antigen of peritoneal exudate cell migration. Conversely, suppression was seen in levels of blood monocytes and in vitro function of macrophages from 6-MP-treated animals in several respects: (a) adherence to glass, (b) migratory rate, (c) phagocytic capacity. Therefore, it would appear that a ma]or mechanism of 6-MP-induced suppression of delayed hypersensitivity is through its action on effector cells.

In vitro lymphocyte reactivity during depression of tuberculin hypersensitivity by 6-mercaptopurine.

Administration of 6-mercaptopurine suppressed appearance of tuberculin skin test reactivity for up to 6 weeks after mycobacterial injection. Lymphocytes obtained during the period of suppressed tuberculin reactivity exhibited normal in vitro proliferative responses to tuberculin, suggesting that the drug may not be qualitatively affecting function of immunologically competent cells.

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.

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.

The late-phase reaction: role of IgE, its receptor and cytokines.

Late phase IgE-mediated reactions (LPR) likely play a major role in the pathogenesis of chronic allergic diseases. Although a number of cellular and humoral alterations in LPR sites have been described recently, the pathogenesis of the LPR is still unclear. LPR follow IgE-mediated but not opiate-induced mast-cell activation suggesting different patterns of mast cell mediator release and/or additional effects of the antigen-IgE interaction. Granulocyte accumulation in developing LPR is accompanied by altered expression of adhesion molecules on local vascular endothelium. Lymphocytes and the cytokines they produce play an important role in developing and perpetuating LPR. These findings point to the likely complex pathogenesis of LPR with implications for the therapy of allergic diseases.

Platelet activating factor increases expression of complement receptors on human neutrophils.

The phospholipid inflammatory mediator platelet activating factor (PAF) has been shown to stimulate certain functions of polymorphonuclear leukocytes (PMN). However, the effect of PAF on surface complement receptors of PMN has not been described. Using monoclonal antibodies and flow cytometry, we have assessed the effects of PAF on surface expression of membrane receptors for C3bi (CR3) and C3b (CR1) in human PMN. PAF (optimal concentration of 1 x 10(-8) M) increased CR3 190% and CR1 174% compared with unstimulated cells at 37 degrees C, while the PAF analogue lyso-PAF had no stimulatory effect. Both CR3 and CR1 responses to PAF reached maximum levels at 15-30 min. PAF effects were comparable to peak effects induced by LTB4 but less than induced by FMLP. A PAF receptor antagonist, SRI 63-441, blocked the increased complement receptor expression in a dose-dependent manner with maximal inhibition of 80-95% at 5 x 10(-6) M. Extracellular calcium had no effect on CR1 expression but slightly enhanced and EGTA partially inhibited the PAF-induced increase in CR3 expression. Simultaneous incubation with PAF and LTB4 enhanced CR3 and CR1 expression more than either agent alone. These findings indicate that PAF, alone and in combination with LTB4, can induce altered expression of complement receptors on the surface of PMN. This effect may enhance adhesion and phagocytosis by PMN at inflammatory reaction sites.

Comparison of cytosolic calcium shifts, superoxide generation, and lactoferrin secretion in the neutrophils of atopics and nonatopics.

We previously found that platelet-activating factor (PAF) stimulated greater lactoferrin secretion by neutrophils of atopics than nonatopics. To help understand underlying mechanisms, we compared agonist-stimulated lactoferrin secretion with another response, superoxide generation, to determine whether there is a global alteration in the reactivity of atopic neutrophils to these stimuli. We also determined Ca2+ mobilization in such neutrophils because such mobilization is a signaling pathway for both superoxide generation and granule content exocytosis. Although PAF again stimulated greater lactoferrin secretion in atopic than in nonatopic neutrophils, the peak superoxide secretion and cytosolic Ca2+ mobilization were not significantly different in atopics and nonatopics. Leukotriene B4-induced superoxide secretion and cytosolic Ca2+ shifts were also similar in atopics and nonatopics. These findings suggest that (1) the enhanced PAF-induced lactoferrin secretion in atopic neutrophils is not a reflection of greater PAF binding or broad-based cell hyperreactivity and (2) a selective signaling pathway in atopic neutrophils may be responsible for the enhanced lactoferrin secretion. These findings may be relevant to in vivo events because we have found increased PAF and lactoferrin release in skin chambers overlying immunoglobulin E-mediated human skin reactions.

Extracellular localization of human connective tissue mast cell granule contents.

In early phases of cutaneous inflammation, connective tissue mast cell degranulation is associated with apparent secretion and externalization of immunoreactive chymotryptic serine proteinase. To determine whether this event is associated with structural evidence of granule externalization, we studied the sequential evolution of IgE-mediated hypersensitivity in vivo, as well as mast cell degranulation provoked by a variety of stimuli in cultured explants of human skin. By 1 min after intradermal antigen challenge with ragweed extract, mast cell degranulation was associated with apparent extrusion of intragranule constituents into the pericellular connective tissue. Similar features typified cultured skin explants exposed for 45 min to anti-IgE and other mast cell secretagogues (morphine sulfate, calcium ionophore A23187, compound 48/80, and substance P). Once externalized, granule constituents could be identified within the dermal matrix by their rounded contour and structural similarity to solubilized granule matrices remaining within actively secreting cells. These data indicate that externalization of connective tissue mast cell granule contents occurs early after secretagogue exposure, potentially accounting for infrequent documentation of this event in naturally occurring dermatoses. The ability to recognize externalized granule products at a morphologic level should facilitate the understanding of interactions between mast cell-derived mediators and target structures of the dermal microvasculature.

Thymic and peripheral blood T- and B-cell levels in myasthenia gravis.

We have compared the percentage of T and B lymphocytes in the thymus and peripheral blood populations of patients with myasthenia gravis. There were significantly fewer thymic T cells in myasthenic hyperplastic thymus (MG-H), but not in myasthenia gravis-thymoma (MG-T), compared with normal thymus biopsies obtained at cardiac surgery. Conversely, B cells were increased in MG-H versus MG-T and normals. Peripheral blood T and B cells were not different in any group of myasthenic patients compared to normal populations. In vitro autologous mixed lymphocyte reactions between thymus and peripheral blood lymphocytes occurred in MG-H, but did not correlate with the degree of thymic B-cell increases in these patients.

Spinal fluid basic protein immunoreactive material and spinal fluid lymphocyte reactivity to basic protein.

Levels of cerebrospinal fluid (CSF) basic protein (BP) immunoreactive material and CSF lymphocyte in vitro reactivity to BP were determined in patients with demyelinating and other inflammatory diseases of the nervous system. Elevated levels of BP and enhanced in vitro reactivity were observed, but there was no correlation between the magnitude of the in vitro response and the amount of BP-like material in CSF.

Immunologic characterization of cerebrospinal fluid lymphocytes: preliminary report.

Cellular immunocompetence of cerebrospinal fluid lymphocytes was investigated in several neurologic diseases. Microtechniques were developed to enable determination of E-rosetting capacity and phytohemagglutinin responsiveness of scant numbers of cells present in the cerebrospinal fluid specimens studied. Although most individuals had phytohemagglutinin-responsive cells in their CSF, reactivity was somewhat less than that found simultaneously in their blood. Three of eight patients had comparable percentages of E rosettes in their blood and CSF. Int the remainder, the values differed significantly. Although preliminary, these result illustrate a new approach to immunologic characterization of CSF lymphocytes in diseases.

In vitro synthesis of antibodies to acetylcholine receptor by peripheral blood mononuclear cells of patients with myasthenia gravis.

We studied the in vitro synthesis of antibodies to acetylcholine receptor (anti-AChR) by peripheral blood mononuclear cells (PBM) of patients with myasthenia gravis (MG) and normal subjects (NS). PBM from three of eight patients with generalized MG (MG-G) synthesized anti-AChR in vitro in the absence of pokeweed mitogen (PWM), and seven of eight did so in the presence of PWM. In individual subjects with MG-G, the levels of anti-AChR secreted in vitro by PBM correlated with serum anti-AChR antibody levels (r = 0.77) but not with the amount of IgG secreted in vitro (r = 0.44). No anti-AChR secretion was seen in culture of PBM from a patient with ocular MG, a patient with thymoma without MG, or six NS.

Cell-mediated immunity to measles, myelin basic protein, and central nervous system extract in multiple sclerosis. A longitudinal study employing direct buffy coat migration inhibition assays.

Cell-mediated immunity to myelin basic protein, to an extract of central nervous system white matter, and to measles virus nuclear core, was studied nine patients with multiple sclerosis in a serial longitudinal fashion using in vitro inhibition of buffy coat migration. The mean migration index to all antigens at various times before, during, and after exacerbation of multiple scelrosis in the patients did not differ from the index in a reference group of normal subjects. The incidence of inhibition of migration induced by central nervous system white matter and basic protein was greater than in serially studied normal subjects (p less than 0.05, p less than 0.2 less than 0.1, respectively) but bore no definite relation to clinical course.

Thymic lymphocyte subpopulations in myasthenia gravis.

We compared the percentages of thymic mononuclear cells (TMC) that bind monoclonal antibodies to T-cell subpopulations in patients with myasthenia gravis (MG) and non-MG patients undergoing cardiac surgery (CS). There were no significant differences in percentages of OKT3+, OKT4+, OKT6+, or OKT8+ cells or the OKT4:OKT8 ratio. There was an increase in the percentage of Ia+ (immune response gene-associated antigen) TMC in MG compared with CS but no significant differences in B cells or phagocytic cells. The Ia+ cells could be abnormal B cells, activated T cells, or thymic dendritic cells.

Immune reactions to P2 protein in human inflammatory demyelinative neuropathies.

The significance of immune reactions against peripheral nervous system antigens in the human inflammatory polyneuropathies is still uncertain. Using a very sensitive assay, we found greatly increased levels of anti-P2 antibodies in sera of animals with experimental allergic neuritis (EAN) but no increases in humans with acute Guillain-Barré syndrome (GBS), chronic relapsing polyneuritis (CRIP), axonal neuropathy, or normals. P2 protein and CNS basic protein did not induce any increased proliferation in lymphocytes of GBS or CRIP patients. We conclude that P2 and BP appear unlikely to be targets for humoral or cellular immune reactivity in GBS or CRIP.

Thymic B-cell activation in myasthenia gravis.

We studied secretion of immunoglobulin (Ig) by freshly isolated and pokeweed mitogen (PWM)-stimulated thymus cells and blood mononuclear cells in patients with myasthenia gravis (MG) and control subjects undergoing elective cardiac surgery. We used a protein A reverse hemolytic plaque assay to enumerate cells secreting IgG, IgM, and IgA (IgSC), and an ELISA assay for measuring IgG secreted into culture supernatants. We found that freshly isolated suspensions of MG thymus cells, compared with control thymus cells, contained increased numbers of cells that spontaneously secreted immunoglobulin. Thymus mononuclear cells from control as well as MG patients appeared capable of B-cell differentiation responses when stimulated by PWM. PWM-induced responses were greater in thymic than in autologous blood mononuclear cells in some MG patients and controls, although B cells were much less frequent in suspensions of thymic cells than blood cells. Thus, the thymus provides a favorable milieu for differentiation of its few B cells. In MG, the thymus may be a site of accentuated in vivo B-cell activation, as evidenced by increased numbers of resident IgSC.

In vitro synthesis of antibodies to acetylcholine receptor by peripheral blood cells: role of suppressor T cells in normal subjects.

Peripheral blood mononuclear cells of 15 of 20 patients with generalized myasthenia gravis synthesized antibodies to acetylcholine receptor (AChR) when the cells were stimulated in vitro with pokeweed mitogen. In contrast, mononuclear cells of 1 of 16 normal subjects synthesized detectable AChR antibodies. Peripheral blood mononuclear cells of five normal subjects were studied before and after putative suppressor T cells (OKT8+) were removed by a fluorescent activated cell sorter. Depletion of OKT8+ cells did not result in production of AChR antibodies, but pokeweed mitogen-induced polyclonal IgG synthesis and activation of B cells to form immunoglobulin-secreting cells (reverse hemolytic plaque assay) were increased. Therefore, failure of blood mononuclear cells of normal subjects to synthesize detectable anti-AChR in response to pokeweed mitogen is not due to suppression by OKT8+ cells.

In vitro cellular responsiveness in multiple sclerosis patients to a viral isolate from multiple sclerosis brain tissue and to other antigens.

The Clausen modification of the leukocyte migration test was used to test patients with multiple sclerosis, normal subjects, and patients with other neurologic diseases for cell-mediated immunity to 6/94 virus (a parainfluenza virus previously isolated from the brain tissue of a multiple sclerosis patient), c-RNA virus isolated from a tumor, and the nonviral antigens Candida and purified protein derivative. Leukocytes of multiple sclerosis patient showed significantly less mean inhibition of migration by the 6/94 virus (but not by the c-RNA virus, purified protein derivative, and Candida) than did the cells of normal controls and patients with other neurologic diseases. The relationship of these findings to previous observations in this area and to the pathogenesis of multiple sclerosis is discussed.

Polyclonal B-cell activity in myasthenia gravis.

Using a protein A-reverse hemolytic plaque assay, we found that some patients with myasthenia gravis have increased numbers of circulating immunoglobulin secreting cells (IgSC). This pattern was not related to drug therapy, age, sex, duration of symptoms, thymectomy, or serum levels of AChR antibody, although elevated IgSC values tended to occur in patients with active symptoms. The responses of peripheral blood mononuclear cells to pokeweed mitogen were normal. These data suggest increased in vivo polyclonal B-cell activation in some myasthenic patients, although in vitro polyclonal B-cell activation is normal.

Analysis of B-cell activation of cerebrospinal fluid lymphocytes in multiple sclerosis.

We have developed a microculture system to study pokeweed mitogen (PWM)-induced B-cell differentiation responses of CSF lymphocytes from patients with multiple sclerosis (MS) and other neurologic diseases (OND). B-cell differentiation was assessed by (1) enumeration of immunoglobulin-secreting cells (IgSC) by a protein A reverse hemolytic plaque assay; and (2) quantitation of supernatant IgG by ELISA. Cultures of MS CSF cells and OND CSF cells responded to PWM with a similar frequency, with responses in CSF cell cultures exceeding responses in corresponding blood cell cultures in several instances in both groups of patients. Numbers of IgSC in unstimulated cultures of MS CSF cells exceeded numbers in cultures of autologous peripheral blood mononuclear cells (PBM). Results suggest that CSF cells may be a particularly reactive population compared with PBM.