Asthmatic patients have neutrophils that exhibit diminished responsiveness to adenosine.

Activation of neutrophils (PMN) within the airways results in the secretion of a number of products such as reduced oxygen metabolites that could contribute to the inflammatory response associated with asthma. However, mediators of allergy, such as histamine, prostaglandin E2 (PGE2), isoproterenol, and adenosine, may serve to mitigate this inflammation through feedback inhibition of neutrophil function. To test the hypothesis that PMN activation and feedback inhibition mechanisms may be abnormal in asthmatics, we compared both superoxide production and adenosine-induced suppression of superoxide production in 12 matched pairs of asthmatics and control subjects. PMN obtained from asthmatic patients generated significantly more superoxide in response to f-met-leu-phe (fMLP) than controls (2.94 +/- 55 nmol/5 x 10(5) PMN/5 min versus 1.38 +/- 0.35 at 2 x 10(-8) M fMLP and 3.81 +/- 0.68 nmol versus 2.04 +/- 0.45 nmol at 10(-7) M; p less than 0.01 for both). In contrast, the respiratory burst generated by two receptor-independent stimuli, the calcium ionophore A23187 and phorbol myristate acetate, was equivalent between control and asthmatic subjects. At 10(-6) M, 2-chloroadenosine induced a 19.5 +/- 5.1% inhibition of fMLP-stimulated superoxide production in PMN from patients with asthma as compared to 55.6 +/- 24.6% inhibition in PMN from control subjects (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Immune mechanisms in allergen-specific immunotherapy.

Allergen-specific immunotherapy has been shown to be clinically effective in patients with seasonal allergic rhinitis and/or asthma. Patients who receive this therapy undergo a number of specific immunologic changes in response to the allergen being administered. These include a "blunting" of the seasonal rise of allergen-specific IgE as well as lowering baseline IgE levels, generation of an allergen-specific IgG response, development of auto-anti-idiotypic antibodies, reduced basophil histamine release in response to allergen, decreased lymphocyte proliferation, lymphokine production in response to allergen, and the generation of allergen-specific suppressor T cells that down-regulate lymphoproliferative responses and IgE synthesis. The mechanism by which allergen-specific immunotherapy produces clinical efficacy is not known. Recent evidence suggests that the development of immunoregulatory responses (suppressor T cells and anti-idiotypic antibodies) during immunotherapy may account for the immunologic changes described above but as yet have not been correlated with clinical outcome. Identification of epitopes on allergens that can induce selective T helper/suppressor responses may provide opportunities for producing immunological tolerance and a reduction in the allergic diathesis.

Dysregulation of arachidonic acid release and metabolism by atopic mononuclear cells.

We studied the ability of monocytes to metabolize [3H]arachidonic acid (AA) provided exogenously by activated T cells, and the extent to which dexamethasone suppressed eicosanoid production by normal and atopic cells. [3H]AA metabolites were identified using a reverse-phase high pressure liquid chromatography system (HPLC). Unstimulated and PHA-stimulated T cells from normal and atopic subjects exhibited a similar uptake and time-dependent release of radiolabel, 90% of which was identified as free AA. The addition of autologous normal and atopic monocytes to these cultures enhanced the release of radiolabel, even in the absence of stimulation with mitogen. Atopic T cell/monocyte cultures released significantly (P = 0.046) more radiolabel than normal cells when stimulated with PHA. Furthermore, the monocytes from both normal and atopic subjects metabolized T cell derived [3H]AA into cyclo-oxygenase (CO) and lipoxygenase (LO) products. Under unstimulated conditions, atopic cells produced significantly (P = 0.04) less CO products than normal cells. In contrast, under PHA and calcium ionophore-stimulated conditions, the atopic cells produced significantly (P = 0.048) more prostaglandins than normal donor cells. Furthermore, although the total release of radioactivity was comparable in both groups, significantly less (P = 0.02) free AA remained in ionophore-stimulated culture supernatants from atopic cells. In order to study the regulation of AA release by normal and atopic T cells, dexamethasone (1 microM) was added to T cell cultures. Dexamethasone inhibited the release of [3H]AA from normal T cells to a significantly (P = 0.003) greater extent than it did to atopic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of IgE synthesis in anti-IgM-activated nonatopic human B cells by recombinant interleukin-3.

The signals required to induce purified normal human B cell subpopulations into IgE production were studied. Pokeweed mitogen (PWM)-stimulated T cell supernatant induced IgE synthesis in low-density but not high-density Percoll-gradient-separated resting B cells. The PWM supernatant also enhanced (greater than 2-fold) IgE synthesis by anti-IgM (but not Staphylococcus A Cowan I)-activated high-density B cells (but not low-density B cells) and had affinity for lentil lectin. Subsequent studies were carried out using purified human recombinant interleukins (rIL). Human rIL-3 consistently (10/19 experiments) augmented IgE synthesis by anti-IgM-activated normal B cells. rIL-4 did not consistently (4/23 experiments) induce IgE synthesis by normal B cells or mixtures of T and B lymphocytes +/- monocytes. Furthermore, IL-1, IL-2, IL-5, IL-6, granulocyte, macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF) and interferon-gamma also failed to induce IgE synthesis. Identification of IL-3 as the factor(s) in the PWM supernatant that was responsible for inducing IgE synthesis was inconclusive since its effect could not be reversed by the addition of anti-IL-3 antibody. Thus, our results suggest that at least two distinct soluble factors are involved in the induction of IgE synthesis by nonatopic B cells.

Leukocyte inhibitory factor stimulates neutrophil-endothelial cell adhesion.

We investigated the ability of the human lymphokine leukocyte inhibitory factor (LIF) to modulate neutrophil-endothelial cell (EC) adherence. EC were cultured from collagenase-treated human umbilical cord veins and grown in complete medium supplemented with EC growth factor. Adherence was measured as the percent of 51Cr-labeled neutrophils remaining adherent to the EC after gentle lavage. Polymorphonuclear neutrophils (PMN) were pretreated with LIF (0.5 to 8 U/ml), extensively washed, and allowed to interact with the EC monolayers. LIF was demonstrated to induce an increase in the capacity of PMN to bind EC in a dose-dependent fashion (from 30.9 +/- 2.1% adherence with control-treated PMN to 68.6 +/- 3.0% at 4 U LIF; p less than 0.001). In subsequent experiments we demonstrated that 10 min was a sufficient preincubation time for LIF to modulate the capacity of the PMN to adhere to EC. LIF has previously been observed to up-regulate expression of C receptor type 3 on PMN, a receptor which has been shown to be involved in PMN-EC binding. Exposure of PMN to anti-C receptor type 3 antibody before their incubation with LIF abrogated its effect as did inactivation of LIF by an esterase inhibitor. We also investigated the ability of LIF to stimulate EC to bind untreated PMN. EC were pretreated with LIF (0.25 to 4 U/ml), extensively washed, and adherence measured as before. LIF was shown to induce a dose-dependent increase in the capacity of the EC to bind PMN (from 28.8 +/- 3.1% for untreated EC to 91.1 +/- 4.0% at 4 U LIF; p less than 0.001). Modulation of EC function required a minimum of 30 min and was inhibited in the presence of cycloheximide or actinomycin D. Neither anti-TNF-alpha or -beta antibodies nor polymixin B abrogated the augmentation by LIF. However, anti-IL-1 antibody partially inhibited the stimulation of EC adhesiveness by LIF, suggesting the possible involvement of this cytokine. These studies provide further evidence that LIF may mediate an important pro-inflammatory role in vivo.

T- and B-cell-derived supernatant factors enhance IgE synthesis by a myeloma cell line (U-266).

IgE synthesis by the human myeloma line U-266 was enhanced 3- to 15-fold in the presence of supernatants from cultures of mononuclear cells (MNC). The enhancing activity was concentration-dependent and was derived from cells that were cultured in the absence of serum and received no in vitro stimulation by exogenous mitogens or lymphokines. T- and B-lymphocyte-enriched populations isolated from MNC were found to generate the enhancing activity, but no enhancing activity was produced by monocytes. MNC from atopic and nonatopic donors were equally effective as sources for this activity. The enhancement of IgE synthesis was proportionally greater than the effect of the activity on cell proliferation. Furthermore, this enhancement of IgE synthesis was demonstrated to be isotype-specific in that the factor(s) had no effect on IgM- and IgG-secreting cell lines. It is suggested that augmentation of IgE synthesis by B cells at a late stage of differentiation may be accomplished by lymphokines constantly present in the cells' milieu and that the U-266 model may be useful for testing putative IgE regulatory factors.

Comparison of prostaglandins E2 and D2 as inhibitors of respiratory burst in neutrophils from atopic and nonatopic subjects.

Neutrophils from atopic and nonatopic donors were treated with prostaglandins D2 and E2 before stimulation of the respiratory burst. Both agents inhibited neutrophil response to formyl-methionyl-leucyl-phenylalanine, but superoxide production was inhibited much more profoundly by D2 than by E2. Inhibition was similar in atopics and nonatopics. Phorbol myristate acetate stimulation of superoxide production was not significantly altered by prostaglandins. These findings suggest that minor alterations in pathways of prostaglandin synthesis may have major effects on modulation of neutrophil function, and exploration of the mechanism of stimulus-specific inhibition may further elucidate the role of neutrophils in the inflammatory response.

Potentiation of neutrophil aggregation by human leukocyte inhibitory factor.

The current studies were designed to extend our investigations on the ability of the lymphokine leukocyte inhibitory factor (LIF) to function as a neutrophil activator. Specifically, we investigated whether LIF could modulate neutrophil (PMN) aggregation. Aggregation was measured as the increase in light transmission using a Payton aggregometer. We found that up to 16 units of LIF was not able to directly induce PMN clumping. However, when preincubated with 0.5-16 units LIF for 10 min, PMN aggregation was significantly enhanced in a dose-dependent manner after stimulation with 10(-7) M FMLP (94.5 +/- 3.1%), 20 nM leukotriene B4 (183.1 +/- 8.2%), and 100 micrograms guinea pig serum-opsonized zymosan (29.8 +/- 8.6%). While the LIF preparation used in these studies was highly purified, specificity for the LIF effect was demonstrated by the ability of several treatments to prevent augmentation of aggregation including: (1) the competitive binding of LIF to one of its substrates: benzoyl-arginine-ethyl-ester; (2) the blocking of PMN LIF receptors with N-acetyl-D-glucosamine; and (3) phenylmethylsulfonylfluoride treatment of the LIF preparation. As aggregation is thought to represent the in vitro correlate to adherence, these studies provide further evidence for a proinflammatory role for LIF in vivo.

Enhancement of the function of neutrophil type-1 and type-3 complement receptors by human leukocyte inhibitory factor (LIF).

The lymphokine leukocyte inhibitory factor (LIF) has previously been documented to enhance several neutrophil (PMN) functions, including stimulated chemotaxis and superoxide generation, phagocytosis and adherence of opsonized targets, and antibody-dependent cellular cytotoxicity. The present studies were designed to investigate the effects of LIF on PMN function mediated by the complement components C3b and C3bi. LIF induced a dose-dependent increase in superoxide production generated by opsonized zymosan (up to 97.1 +/- 31.4% at 16 U LIF/ml; P less than 0.01). While neither control nor LIF-treated PMN were capable of inducing phagocytosis of either C3b- or C3bi-opsonized sheep erythrocytes (E) directly, exposure to LIF caused a significant (P less than 0.05) increase in their adherence to E (137.4 and 59.4%, respectively). Specificity for complement receptor function was confirmed by the ability of anti-CR1 antibody to block adherence of LIF-treated PMN to EAC3b (77.0% inhibition) and anti-CR3 antibody to block adherence to EAC3bi (70.2% inhibition). Increased C3b and C3bi function may have been due, at least in part, to increased expression of their respective surface membrane receptors. Thus, using indirect immunofluorescence, LIF induced a 38.2% increase in fluorescence of the anti-CR1 antibody and a 96.1% increase in anti-CR3 binding. These studies describe an additional mechanism through which LIF may have an important pro-inflammatory role in vivo.

Double-blind comparison of terfenadine, chlorpheniramine, and placebo in the treatment of chronic idiopathic urticaria.

The efficacy of terfenadine, a nonsedating H1 antihistamine, in the management of chronic idiopathic urticaria was compared with chlorpheniramine and placebo in a parallel multicenter trial. Subjects with symptoms of hives for 3 days per week for at least 6 weeks were initially screened and admitted if no identifiable cause for symptoms could be determined. Patients entered a single-blind placebo period, and if hives of moderate severity were present for at least 3 days during the week, they were randomly assigned in a double-blind fashion to take terfenadine, 60 mg twice daily, chlorpheniramine, 4 mg three times a day, or placebo for 6 weeks. Data were analyzed for 122 patients. Those patients receiving both active treatments noted significant improvement in symptoms: pruritus, redness, number of hives, and waking hours during which hives were present, at the end of the first day of therapy. Symptom control by terfenadine was statistically superior to placebo during all 6 weeks, as rated by both patients and investigators. However, statistical significance was not achieved for chlorpheniramine at all observation points. Diphenhydramine was permitted as a relief medication for refractory symptoms and was taken by 52% of subjects receiving placebo, 26% taking chlorpheniramine, and only 9% of patients who were receiving terfenadine. In addition to providing superior symptom control, terfenadine caused less drowsiness and fatigue than chlorpheniramine. Terfenadine is a useful therapeutic agent for primary management of chronic idiopathic urticaria.

Characterization of the neutrophil respiratory burst in atopy.

Activated neutrophils may play a part in atopic disorders. In these studies, neutrophils were obtained from atopic and nonatopic adults for assessment of respiratory-burst activity. Superoxide production was measured in the resting state and after stimulation with phorbol myristate acetate, formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe), or calcium ionophore A23187. Inhibition of this response by histamine was also measured. Neutrophils from atopic subjects produced more superoxide in the basal state, or in response to calcium ionophore A23187, or submaximal concentrations of f-met-leu-phe, than cells from control subjects. Histamine inhibition of f-met-leu-phe-stimulated superoxide production was less in atopic subjects than in control subjects. These findings suggest that neutrophils from atopic subjects may be hyperreactive in that they produce more superoxide in response to stimuli of low potency and are less readily inhibited than cells from control subjects. These properties may reflect a general attribute of cellular function in atopy and may contribute directly to tissue damage in allergic diseases.

Detection of human auto-anti-idiotypic antibodies (Ab2). I. Isolation and characterization of Ab2 in the serum of a ragweed immunotherapy-treated patient.

This study documents, by means of both solid and liquid phase assays, the presence of anti-idiotypic antibodies (Ab2) in the serum of one allergic individual undergoing ragweed immunotherapy. Serum from this individual was collected and F(ab')2 fragments specific for ragweed antigen E (AgE) were prepared (Ab1). These AgE-specific F(ab')2 Ab1, following absorption with normal immunoglobulin, were studied for their capacity to specifically bind autologous Ab2. The binding of Ab1 to Ab2 was demonstrated to be inhibitable by ragweed AgE but not by another allergen (oak) to which the patient reacted. These findings demonstrate the presence and binding specificities of Ab2 in the serum of a ragweed immunotherapy patient and may serve as a model to study the role of Ab2 in the regulation of Ab1 in allergic patients.

Detection of human auto-anti-idiotypic antibodies (Ab2). II. Generation of Ab2 in atopic patients undergoing allergen immunotherapy.

The present study demonstrates the cross-reactivity of a murine monoclonal antibody (MoAb) directed against purified ragweed antigen E (AgE) with human Ab1. This antiragweed AgE MoAb (clone SC7H.1G, IgM kappa) was used to detect Ab2 in the sera obtained from the three groups of subjects. Utilizing an ELISA assay, we found that immunoglobulins from 12 nonatopic subjects bound to a significantly greater extent to SC7H.1G (mean +/- SE; OD = 0.353 +/- 0.052) than immunoglobulins from 14 untreated ragweed atopics (OD = 0.149 +/- 0.020). However, immunoglobulins from 9 immunotherapy-treated patients (OD = 0.395 +/- 0.120) bound to the mouse anti-AgE MoAb to the same extent as nonatopic sera. Furthermore, an additional 7 patients with ragweed-allergic rhinitis were studied prospectively while undergoing immunotherapy and Ab2 levels were found to increase with time posttreatment. We also found an inverse correlation between Ab1 and Ab2 levels in the nonatopic and untreated atopic groups. These data indicate that immunotherapy stimulates the production of Ab2 in atopic patients and may be involved in the regulation of the antiragweed IgE response.

Reproductive immunology.

The maternal immune system is challenged with paternal antigens through exposure to trophoblast tissue and fetal cells crossing the placenta into the maternal circulation. The dose of antigen, the manner of presentation (cellular, subcellular, or soluble), and the nature of the antigen all determine the type of response that will be elicited. It is also clear that complex maternal immunologic responses, including antibodies to red blood cell antigens, HLA-A, HLA-B, HLA-C, and HLA-D antigens, and cell-mediated responses such as proliferation, lymphokines, cytotoxicity, and suppressor cells, are generated to a variety of paternal antigenic determinants. The fact that some of these reactions are detected in vitro in the absence of maternal serum, but not in its presence, suggests that the local milieu is important in influencing their expression in vivo. For example, such factors as hormones (cortisol, progesterone, and estrogen), pregnancy-associated glycoproteins (alpha 2-macroglobulin and beta 1-glycoprotein) and AFP, which have immunosuppressive properties, may all serve nonspecifically to inhibit and decrease the general tone of maternal immunologic responses, particularly at the placental interface, where many of these factors are present in high concentrations. However, these nonspecific factors may not be sufficient to prevent presensitized effector lymphocytes from continuing an ongoing rejection process, as is often the case in the chronic rejection of an allograft. For this purpose, specific enhancing antibodies would play an important role by blocking maternal responses or protecting the fetus. There may be a subtle balance created on the trophoblast cell surface between specific antibodies and trophoblast or embryonic alloantigens, resulting in limited expression of antigens capable of inducing rejection reactions. This could favor the production of blocking antibodies and/or T-suppressor cells, as opposed to cytotoxic antibody and killer cells. In fact, low levels of antigen density on the cell surface favor a blocking effect by IgG rather than cytotoxicity. Blocking or enhancing antibodies can exert their effect on maternal immunologic responses in several ways. They could block the afferent limb by combining with antigen and preventing sensitization or increasing the level of sensitivity. An example of the latter would be the coating of fetal cells that enter the maternal circulation. Enhancing antibodies could work directly on the effector cells to suppress their function. The antibody itself, or more likely antigen-antibody complexes, may be important in this regard.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphorylation and dephosphorylation of soluble proteins in human eosinophils.

The effect of phorbol 12-myristate 13-acetate (PMA), calcium ionophore (A23187), opsonized zymosan (OZ), and N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) on protein phosphorylation was examined in purified eosinophils (eos) isolated from human peripheral blood. Eos were prelabeled with [32P]orthophosphate, stimulated with several activating agents for varying periods of time. The soluble proteins were then analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. In resting eos, there was phosphorylation of endogenous soluble proteins with molecular weights of 12, 16, 21, 40, and 66 kilodaltons (kDa). PMA, a potent activator of oxidative metabolism, induced phosphorylation of 19-, 40-, and 67-kDa proteins. A23187, a strong degranulating stimulus, caused phosphorylation of 40-, 53-, and 67-kDa proteins. OZ, a relatively weak stimulus for eos function, caused phosphorylation of 30-34-, 59-, 67-, and 93-kDa proteins. In addition, all the above stimuli caused a time-dependent dephosphorylation of 21-kDa protein. In contrast, f-Met-Leu-Phe caused neither phosphorylation of new proteins nor dephosphorylation of preexisting eos proteins. These findings demonstrate that selected stimuli affect phosphorylation of soluble eos protein. These results also suggest that phosphorylation of specific proteins in eos is an intermediary step in external stimulus-induced cell activation, which may involve many different cell functions.

Generation of suppressor cells in atopic patients during immunotherapy that modulate IgE synthesis.

We prospectively studied the effect of ragweed immunotherapy on the generation of suppressor T cells that modulate total and antiragweed IgE production in five patients with seasonal allergic rhinitis. Suppressor T cell depletion was accomplished by treating mononuclear cells (MNCs) with the monoclonal antibody Leu 2b followed by the addition of complement. Treated and untreated MNCs were obtained before and during (6 to 24 months) immunotherapy and were cultured at 1 X 10(6) cells per milliliter for 7 days in RPM1-1640 containing 10% fetal calf serum. In order to determine the effect of ragweed antigen on IgE production, untreated or Leu 2b-depleted MNCs were incubated with short ragweed extract (SRW) (1.0 to 10 micrograms/ml) for 20 hours, washed three times, and incubated for a further 6 days. The cell-free supernatants from each were harvested and assayed for total IgE by use of a modified PRIST assay. To determine specific IgE, a modified RAST procedure was used. Total or antiragweed IgE production was calculated by subtracting preformed IgE from the total or specific IgE content in the supernatant. Depletion of Leu 2-positive cells did not affect total IgE production before immunotherapy. In contrast, a statistically significant increase was observed in total IgE 6 months (p less than 0.05) and 13 months (p = 0.005) after immunotherapy. Specific antiragweed IgE production at these times was also enhanced, but this increase did not reach statistical significance. Before immunotherapy was begun, preincubation of MNCs with SRW did not change the amount of total IgE produced.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoid-induced enhancement of IgE synthesis.

Glucocorticoid-induced enhancement of polyclonal immunoglobulin production of the IgG, A, and M classes by human peripheral blood mononuclear cells has been shown to be dependent on regulatory T-cells. The following in vitro investigation demonstrated an enhancing effect of glucocorticoid on IgE production by mononuclear cells from atopic patients, but not by cells from non-atopic subjects. Augmentation was noted even in cell populations depleted of T cells and monocytes. These observations suggest that the corticosteroid acted directly on an IgE B cell at a late stage of differentiation that was no longer dependent on helper T-cells, although an additional effect on T-suppressor cells was not excluded.

Effects of leukocyte inhibitory factor (LIF) on neutrophil phagocytosis and bactericidal activity.

Human leukocyte inhibitory factor (LIF) is a lymphokine initially defined by its ability to inhibit the random migration of neutrophils. We have recently demonstrated that LIF also potentiates a number of f-met-leu-phe-mediated functions as well as enhancing one Fc receptor-mediated function (antibody-dependent cellular cytotoxicity). In this paper, we have extended our studies involving the effects of LIF on the neutrophil, specifically its effect on phagocytosis and bactericidal activity. We demonstrate that LIF (2 U/ml) potentiates phagocytosis of opsonized heat-killed Staphylococcus aureus (up to 57.2%) and sheep erythrocytes (124.4%) as well as unopsonized latex particles (59.9%). Phagocytosis of opsonized sheep erythrocytes was inhibited by an anti-neutrophil Fc receptor antibody with control PMN but not using the LIF-treated PMN. LIF (1/2 to 1 U) also potentiates the killing of S. aureus by up to 51.6%. Higher concentrations of LIF (greater than or equal to 4 U) inhibits killing. These effects were shown not to be associated with an increase in Fc receptor availability. It is therefore possible that potentiation of these neutrophil activities by LIF may occur either as a result of increased receptor turnover or, more likely, secondary to an increase in nonspecific neutrophil adherence. These studies further support the concept that LIF may have an important role in vivo in inflammation and immunity.