TGF-ß-mediated airway tolerance to allergens induced by peptide-based immunomodulatory mucosal vaccination.

We sought to modulate mucosal immune responses using neonatal vaccination to avert the development of allergic airways disease (AAD). Pulmonary pathology in AAD is driven by T helper (TH)2 cytokines, in particular interleukin (IL)4 and IL13, the expression and actions of which are regulated by the transcription factor STAT6. We developed a peptide homolog of STAT6, STAT6-IP. Neonatal mice given, intranasally, STAT6-IP, in an effort to modulate de novo airways immune responses, developed tolerance following subsequent allergen sensitization, with either ovalbumin or ragweed allergens, as demonstrated by reduced TH2 cytokines and specific immunoglobulin (Ig)E and the significant increases in the latency-associated peptide (LAP)(+) T-regulatory (Treg) cell subset and expression of transforming growth factor (TGF)-ß. This regulatory phenotype was transferrable by CD4(+) T cells or CD11c(+) dendritic cells (DCs) derived from STAT6-IP-vaccinated mice. Anti-TGF-ß treatment during allergen sensitization, however, re-established the pro-inflammatory TH2 response. Thus, neonatal STAT6-IP vaccination induces prospective TGF-ß-dependent tolerance to allergen and constitutes a novel highly effective immunomodulatory allergy prevention strategy.

Intravenous immunoglobulin attenuates airway hyperresponsiveness in a murine model of allergic asthma.

BACKGROUND: Intravenous immunoglobulin (IVIG) has potent anti-inflammatory and immune-modulating properties. IVIG has been utilized as a steroid-sparing agent in severe asthma, but the results of clinical trials have been conflicting. OBJECTIVE: To determine whether IVIG is able to attenuate bronchial reactivity, pulmonary inflammation and T cell function using a murine model of allergic airways disease. METHODS: BALB/c or C57BL/6 mice were sensitized to ovalbumin (OVA) or a phosphate-buffered saline control using local nasal sensitization, and then received five intranasal challenges on days 28-32 before sacrifice. Mice were treated intraperitoneally with either IVIG (1-2 g/kg) or equivalent human serum albumin 24 h before the first OVA challenge. Bronchial reactivity to methacholine was examined using the FlexiVent small animal ventilator. We evaluated pulmonary histology, mRNA from lung digests for T-helper type 2 (Th2)-related genes and bronchoalveolar lavage for cell counts and cytokines. Splenocytes were utilized to study OVA-induced cell proliferation, cytokine production and dendritic cell maturation. RESULTS: IVIG markedly attenuated the perivascular and peribronchial pulmonary inflammation, and decreased bronchial hyperresponsiveness to methacholine. IVIG treatment of splenocytes from sensitized animals diminished cellular proliferation to OVA, whereas IVIG treatment in vivo markedly attenuated OVA-driven splenocyte proliferation. This is accompanied by diminished IL-13 and TNF-α levels in splenocyte culture, decreased expression of Jagged-1, increased Delta-4 and decreased GATA-3 mRNA levels, signs that IVIG has suppressed the expected Th2 response that accompanies repeated allergen exposure. Increased regulatory T cells were found in draining pulmonary lymph nodes in IVIG-treated mice but not in controls. CONCLUSIONS AND CLINICAL RELEVANCE: IVIG was effective in ameliorating allergic airway disease in our model. IVIG may be a promising adjunct therapy requiring further study for patients with severe asthma.

Children with atopic histories exhibit impaired lipopolysaccharide-induced Toll-like receptor-4 signalling in peripheral monocytes.

BACKGROUND: The hygiene hypothesis states that early exposure to bacterial products such as lipopolysaccharide (LPS) may be protective against the development of allergic diseases. Whether atopic disease affects the ability of immune cells to respond to LPS is unclear. Our laboratory has demonstrated previously that children express high levels of Toll-like receptor (TLR)-4 on CD4(+) cells in nasal mucosa. OBJECTIVE: To determine if children with a history of allergic disease have impaired responses to LPS on circulating CD4(+) leucocytes. METHODS: Peripheral blood mononuclear cells from children (aged 2-18) and adults with or without a history of atopic conditions were cultured with/without IL-4 or LPS for up to 24 h. Expression of surface TLR-4, CD14, CD4, CD3, as well as of intracellular phosphorylated (p42/p44) ERK and p38 mitogen-activated protein kinase (MAPK) were assessed by flow cytometry. RESULTS: A history of atopy in children was associated with impaired LPS-induced TLR-4-dependent phosphorylation of (p42/44) ERK and p38 MAPK by CD4(+) monocytes. Decreased LPS signalling was reproduced by pre-incubation of control cells with recombinant IL-4. LPS stimulation also decreased TLR-4 expression on monocytes from children without atopic histories but not from atopic subjects. CD4(+) T lymphocytes showed limited LPS responsiveness, regardless of atopic status. In contrast with non-atopic children, TLR-4 expression on monocytes of children with atopic histories decreased as a function of age. CONCLUSIONS: This study provides evidence for defective LPS recognition on circulating CD4(+) leucocytes of subjects with atopic histories compared with those from non-atopic children. CD4(+) TLR4(+) monocytes from children with atopic histories failed to phosphorylate MAPKs. Our results suggest that a history of atopic disease is associated with impaired TLR-4-mediated innate immune function compared with non-atopic children.

B lymphocytes in inflammatory airway diseases.

B lymphocytes are key players in all facets of adaptive immune responses and are responsible for the production of IgE antibodies, initiators of allergic hypersensitivity reactions. Recent evidence indicates that B cells may be a crucial player in allergic and inflammatory airway pathology, directly populating upper and lower airway tissues. This review examines human and animal studies that directly demonstrated the presence of B lymphocytes in airway tissues and elaborates on their function as antibody-secreting cells, antigen-presenting cells and producers of inflammatory and regulatory cytokines. B lymphocytes appear to contribute to multiple facets of immune homeostasis in inflammatory diseases of the upper and lower airways.

Efficient purification of eosinophils from human tissues: a comparative study.

BACKGROUND: Eosinophils are key effector cells in allergy and in other inflammatory diseases. Although they carry out their function in the tissues, no efficient method exists allowing for consistent purification of tissue eosinophils for culture. Rather, studies rely mainly on peripheral blood eosinophils. This study aimed to determine the most efficient protocol for purifying eosinophils from nasal polyp tissue. METHODS: Nasal polyps were obtained from patients undergoing surgical polypectomy. The polyps were minced and enzymatically digested. Surface receptor analysis was performed by flow cytometry. In order to obtain optimal purification, the nasal polyp cell suspension was subjected to two methods of purification: 1) positive magnetic selection of CCR3+cells, or 2) negative selection using CD3/CD14/CD16 magnetic beads. Enriched tissue eosinophils were cultured with or without IL-3, IL-5 or GM-CSF, and their survival was evaluated by flow cytometry. RESULTS: Tissue-derived eosinophils exhibited surface expression of NEC2, DNAM-1, NTBa, 2B4, and CD300a comparable to similarly prepared eosinophils obtained from the peripheral blood of the same patients. Positive selection consistently yielded eosinophils of high purity (>90%) with 63% viability. In contrast, negative selection yielded better viability (88%), reduced purity (66%), and could be utilized for in vitro activation experiments. CONCLUSION: Eosinophils can be purified from nasal polyps. Negative selection appears to be advantageous due to improved viability of the eosinophils, which may be cultured and activated in vitro. This methodology is an important advance in studying tissue eosinophils for further investigations on inflammatory tissue responses.

Signal transducer and activator of transcription 6 down-regulates toll-like receptor-4 expression of a monocytic cell line.

BACKGROUND: Toll-like receptor 4 (TLR4), part of the bacterial lipopolysaccharide (LPS) receptor, is an important bridge between innate and adaptive immunity. Our previous studies have indicated reduced expression of TLR4 and reduced responsiveness to LPS in nasal mucosa of atopic adults compared with non-atopic adults. IL-4 and signal transducer and activator of transcription 6 (STAT6), which are increased in atopic patients, may have a role in modulating TLR4. OBJECTIVE: To examine direct effects of IL-4 and STAT6 on TLR4 expression of U-937 monocytic cells. METHODS: LPS responsiveness, under different conditions of U-937 cells was measured by nuclear factor (NF)-kappaB activation of transcription. TLR4 mRNA was quantified by real-time PCR and TLR4 surface expression was measured by flow cytometry. The promoter and 4.3 kb of the upstream region of TLR4 were cloned into a plasmid vector and transiently transfected into U-937 cells. Transfected cells were incubated with IL-4 and transcriptional activity was assayed by the luciferase assay. STAT6 was transfected to evaluate overexpression of this transcription factor. Cells were also incubated with Tyrphostin AG490 to inhibit tyrosine kinases. RESULTS: NF-kappaB activation by LPS was inhibited by IL-4 pre-incubation but not when IL-4 was added at the same time as LPS. TLR4 mRNA expression was inhibited by IL-4 as early as 6 h but the effect was lost by 24 h. Surface expression of TLR4 was inhibited by IL-4 at 12 and 24 h, but returned to baseline at 48 h. IL-4 inhibited activity of the TLR4 promoter as early as 6 h, but, like the mRNA, these effects were transient. STAT6 overexpression enhanced the inhibition of the TLR4 promoter and prolonged it. Inhibition of TLR4 by IL-4 was abolished by pre-incubation with the tyrosine kinase inhibitor Tyrphostin AG490. CONCLUSION: Our findings demonstrate that IL-4, through STAT6, can modulate TLR4 expression and suggests that Th2 cytokines can impact on the LPS responsiveness of cells.

Functional expression of IL-12 receptor by human eosinophils: IL-12 promotes eosinophil apoptosis.

In murine models of allergic inflammation, IL-12 has been shown to decrease tissue eosinophilia, but the underlying mechanisms are not known. We evaluated the expression of IL-12R and the effect of IL-12 on eosinophil survival. In situ hybridization demonstrated the presence of mRNA and immunoreactivity for IL-12Rbeta1 and -beta2 subunits in human peripheral blood eosinophils. Surface expression of IL-12Rbeta1 and -beta2 subunits on freshly isolated human eosinophils was optimally expressed after incubation with PMA. To determine the functional significance of IL-12R studies, we studied cell viability and apoptosis. Morphological analysis and propidium iodide staining for cell cycle demonstrated that recombinant human IL-12 increased in vitro human eosinophil apoptosis in a dose-dependent manner. Addition of IL-5 together with IL-12 abrogated eosinophil apoptosis, suggesting that IL-12 and IL-5 have antagonistic effects. Our findings provide evidence for a novel role for IL-12 in regulating eosinophil function by increasing eosinophil apoptosis.

Activation via multiple signaling pathways induces down-regulation of platelet-activating factor receptors on human B lymphocytes.

Platelet-activating factor receptor (PAFR) has been identified in B cell lines and primary human B cells, but the regulation of PAFR during B cell activation has not been completely elucidated. In the present study, we have investigated the effects of B cell activation on PAFR binding parameters, PAFR mRNA and PAF-triggered intracellular calcium mobilization. The human B lymphoid cell line LA350 was shown to exhibit high levels of PAFR (48,550 +/- 4,310 sites/cell) as determined by radio-ligand binding assay with PAFR antagonist [3H]WEB2086. Treatment with phorbol 12,13-dibutyrate caused a biphasic reduction of PAFR binding. The early phase was inhibited by the protein kinase C inhibitor bisindolylmaleimide I (BIM), whereas the late phase was not blocked by BIM, protein tyrosine kinase inhibitor genistein, or the mitogen-activated protein kinase/extracellular signal-related kinase inhibitor PD98059. However, staurosporine, a broad-spectrum protein kinase inhibitor, completely inhibited the late phase down-regulation. Ionomycin also decreased [3H]WEB2086 binding sites, whereas the combination of PDB and ionomycin induced a greater reduction than either agent alone. Cross-linking of B cell receptor by anti-IgM Ab also induced down-regulation of PAFR, which was abolished by genistein or PD98059, but not by BIM or staurosporine. The decrease in surface PAFR number was closely paralleled by the reduction in PAFR mRNA both in LA350 cells and human tonsillar B cells, and was associated with decreased response to PAF indicated by decreased intracellular calcium mobilization. These data show that multiple signaling pathways are involved in down-regulating PAFR expression during B cell activation and development.

Detection of functional platelet-activating factor receptors on human tonsillar B lymphocytes.

Although platelet-activating factor (PAF) receptors have been found on B lymphoblastoid cell lines, the action of PAF on freshly isolated human B cells has not been clearly demonstrated. Using a sensitive semiquantitative reverse-transcriptase PCR, we have found PAF receptor mRNA expressed by tonsillar B lymphocytes, but little in T lymphocytes. Examination of Percoll-fractionated tonsillar B cells indicated that the low density (primarily germinal center cells) and medium density fractions had approximately twofold more PAF receptor mRNA relative to the high density fraction. PAF (10-7 M) stimulated increases in intracellular Ca2+ that were consistently higher in the low and medium density B lymphocytes compared with high density cells. The PAF receptor antagonist Web 2170 inhibited this. Addition of PAF, but not lyso- or enantio-PAF, induced four- to sixfold greater synthesis of IgM and IgG in low and medium density cells compared with unstimulated controls, but had little effect on Ig production by high density cells. To investigate how PAF may influence Ig synthesis, PAF-stimulated B cells were examined for production of the Th2-type cytokines IL-4 and IL-13. PAF induced IL-4 and IL-13 mRNA expression in 17% of CD20+ cells, and IL-4 was detected in cell supernatants after 48-72 h of culture. Together, these data strongly suggest that functional PAF receptors are expressed on B cells in tonsils.

Dose-dependent agonist and antagonist effects of the platelet-activating factor analogue 1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine on B lymphocytes.

BACKGROUND: Platelet activating-factor (PAF), an ether-linked phospholipid, is a potent activator of B lymphocyte cell lines. The related ester-linked phospholipid, 1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine (PAGPC), is synthesized by tissues important in B-cell development. OBJECTIVES: We examined whether PAGPC was capable of influencing immunoglobulin synthesis in B lymphocytes and compared its action with that of PAF. We also examined the interaction of the two mediators as agonists or competitive antagonists. METHODS: Ramos, an IgM-secreting immature B-cell line that expresses PAF receptor, was used in these experiments. The effect of PAF, PAGPC, or both mediators together on IgM secretion and anti-IgM-mediated apoptosis was measured. RESULTS: Both PAF and PAGPC stimulated IgM production in Ramos cells in a dose-dependent fashion, with PAGPC being approximately three logs less potent than PAF. The effect of both mediators was inhibited by specific PAF receptor antagonists. Preincubation with suboptimal concentrations of PAGPC inhibited the ability of PAF to increase IgM secretion by Ramos cells. Additionally, preincubation with low concentrations of PAGPC prevented PAF from rescuing Ramos cells from apoptosis induced by cross-linking the B-cell receptor with anti-IgM antibodies. PAGPC caused PAF receptor desensitization because displacement of bound PAGPC with high concentrations of bovine serum albumin did not reverse its PAF antagonist effect. CONCLUSIONS: PAF and PAGPC are biologically active phospholipids, but PAF is approximately 1000 times more potent. At high concentrations, PAGPC acts similarly to PAF, whereas at lower concentrations, PAGPC acts as a functional PAF antagonist. Because it is secreted at sites of inflammation and allergic reactions, PAGPC may be an endogenous regulator of the effects of PAF.

Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes.

Recently, a severe combined immunodeficiency syndrome with a deficiency of CD8+ peripheral T cells and a TCR signal transduction defect in peripheral CD4+ T cells was associated with mutations in ZAP-70. Since TCR signaling is required in developmental decisions resulting in mature CD4 (and CD8) T cells, the presence of peripheral CD4+ T cells expressing TCRs incapable of signaling in these patients is paradoxical. Here, we show that the TCRs on thymocytes, but not peripheral T cells, from a ZAP-70-deficient patient are capable of signaling. Moreover, the TCR on a thymocyte line derived from this patient can signal, and the homologous kinase Syk is present at high levels and is tyrosine phosphorylated after TCR stimulation. Thus, Syk may compensate for the loss of ZAP-70 and account for the thymic selection of at least a subset of T cells (CD4+) in ZAP-70-deficient patients.

Role for Ca2+ in expression of cell cycle regulated genes in PAF-stimulated cells.

Platelet-activating factor (PAF) is a powerful stimulator of a wide variety of cells. In transformed human B-lymphoblastoid cell lines, PAF increases intracellular Ca2+ concentrations ([Ca2+]i) and induces the expression of the proto-oncogenes c-fos and early growth response gene-2 (EGR2). Here, we present data that evaluates the role of Ca2+ in the PAF-dependent induction of these cell-cycle activated genes. PAF (10(-7) M) increased c-fos and EGR2 mRNA levels in cells suspended in Ca(2+)-containing medium by 6-10-fold. In PAF-stimulated cells suspended in medium depleted of Ca2+, eliminating Ca2+ influx but not intracellular store release of Ca2+, the induction of gene expression was reduced by approx. 50%. In contrast, buffering of Ca2+ released from intracellular stores but maintaining transmembrane Ca2+ uptake had little effect on gene expression. When both sources of Ca2+ were eliminated, PAF-stimulated expression of these genes was completely prevented. This was not due to any toxicity to the cells since the response to phorbol ester under identical conditions was unaffected. The regulation of c-fos mRNA expression was paralleled by changes in levels of FOS protein. These data indicate that changes in [Ca2+]i, primarily from stimulated entry across the plasma membrane and to a lesser extent release of Ca2+ from sequestered intracellular stores, play an essential role in PAF-dependent triggering of c-fos and EGR2 mRNA expression.

Detection of the human platelet-activating factor receptor mRNA in human B lymphocytes by polymerase chain reaction on reverse transcripts (RT-PCR)

The platelet activating factor receptor (hPAFR) is a GTP binding protein linked receptor of the rhodopsin family. The hPAFR gene maps to chromosome 1 and is characterized by the absence of introns in its coding region. Because PAF demonstrates unique properties as a growth factor in human B lymphoblastoid cell lines, we developed an RT-PCR in order to detect the presence of hPAFR mRNA. We examined the presence of the hPAFR in a series of B lymphoblastoid cell lines, as well as on fresh human B cells and the T-leukemia cell line Jurkat. All cells of B lineage expressed hPAFR mRNA, including a B cell line not previously shown to express functional hPAFR. In contrast, the T-leukemia cell line Jurkat expressed very low levels of hPAFR mRNA. We discuss the methodology and its validation in developing an RT-PCR for intronless genes such as the hPAFR.

Autocrine stimulation of B lymphocytes by a platelet-activating factor receptor agonist, 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine.

Based on previous data which demonstrated the ability of platelet-activating factor (PAF) antagonists to inhibit constitutive immunoglobulin synthesis in B-lymphoblastoid cell lines, we determined the capacity of these cells to synthesize PAF or 1-palmitoyl-2-acetyl-sn-glycero-phosphocholine (PAGPC), an acyl-PAF identified in various cell types. In two B-lymphoblastoid cell lines (LA350 and HSCE-), significant amounts of production of PAGPC were detected, whereas the amount of PAF was below the level of detection in our system. The biologic effects of PAGPC were examined in these cells, both of which have well-characterized PAF receptors. PAGPC induced a concentration-dependent increase in intracellular Ca2+ concentrations and activation of MAP-2 kinase (as detected by immunoblotting and measurements of kinase activity) in these cells. The kinetics and magnitude of these responses were similar to those induced by PAF, and they were inhibited by Web 2086, a PAFR antagonist. Phosphatidylcholine, which differs from PAGPC in that it contains a long fatty acid residue at position 2, did not induce any of these responses. A mutual cross-desensitization of the B lymphoblasts between PAGPC and PAF was observed for Ca2+ mobilization. To induce maximal cell stimulation, approximately 600-fold higher concentrations of PAGPC than of PAF were needed. Because the two B-lymphoblastoid cell lines synthesized significant amounts of PAGPC, this phospholipid may participate in an autocrine stimulation pathway in B cells. Furthermore, the data indicate that PAGPC is an agonist for the PAFR in B lymphoblasts and that the ether linkage in the PAF molecule is not an absolute requirement for activity, although it increases the potency of the ligand.

Platelet-activating factor-mediated transmembrane signaling in human B lymphocytes is regulated through a pertussis- and cholera toxin-sensitive pathway.

Platelet-activating factor (PAF) stimulates human B cells, resulting in elevation of intracellular calcium and the release of inositol phosphates. This signaling pathway is inhibited in the presence of pertussis (PT) or cholera toxin (CT). Preincubation of human B cells with either toxin, but not their inactive subunits, for 3 h blocked these PAF-induced responses in two B-lymphoblastoid cell lines. This effect was time dependent, with some inhibition noted at 30 min, but only after preincubation for 2-3 h was maximum inhibition achieved. This inhibitory activity was also dose dependent. The toxins blocked both PAF-induced transmembrane uptake of Ca2+ as well as release of Ca2+ from internal stores, and were selective in that activation events after cross-linking of surface IgM were not affected. Further, the toxins did not appear to act through elevation of intracellular levels of cAMP. These data, coupled with previous observations on the absence of heterologous desensitization between PAF and sIgM receptors, may delineate distinct signaling pathways in human B cells. This may reflect different roles for GTP-binding proteins in the activation of human B cells.

Ethanol inhibits ligand-activated Ca2+ channels in human B lymphocytes.

Ethanol reportedly is immunosuppressive, interfering with lymphocyte proliferation. To investigate the basis for this immunosuppression, the effects of acute treatment with ethanol were studied on Ca2+ mobilization in tonsillar B lymphocytes and the human lymphoblastoid B-cell line, Ramos. The level of intracellular Ca2+ was monitored in cells loaded with the fluorescent dye indo-1 following stimulation with either anti-IgM antibody or platelet activating factor. The effect of ethanol was also examined on the induction of the early proto-oncogene c-fos in these cells. Ethanol inhibited ligand-activated Ca2+ mobilization due to transmembrane influx but not intracellular store release, in a dose- and time-dependent manner. This inhibition was not due to the inability of anti-IgM to bind to its surface receptor nor to membrane depolarization induced by ethanol. Ethanol also inhibited the Ca2(+)-dependent induction by anti-IgM of c-fos in these cells. The inhibitory effects of ethanol on ligand-activated Ca2+ channels and subsequent induction of c-fos may provide the basis for its immunosuppressive action.

B-cell activation and regulation of immunoglobulin synthesis by platelet activating factor.

Platelet activating factor (PAF) is a highly potent phospholipid mediator known to be active in many biologic systems. To date little is known of the effect of PAF on B lymphocytes. Using three immunoglobulin (Ig)-secreting B-lymphoblastoid lines, we have demonstrated that PAF can influence both early activation and late differentiation events. Following addition of 10(-7) to 10(-11) M PAF, but not the inactive metabolite lyso-PAF, all three cell lines demonstrated rapid, dose-dependent increases in free cytosolic Ca2+ concentrations ([Ca2+]i). The changes in [Ca2+]i resulted from release of intracellular stored Ca2+ as well as transmembrane Ca2+ uptake. In parallel PAF caused significant alterations in the kinetics of Ig secretion in the Ig-secreting lymphocyte lines. A 6-12-fold increase in Ig production was detectable after 24 h of stimulation with PAF, which was followed by a plateau over the next 48 h. All of these events were inhibited by the specific PAF antagonists Web2086 and CV3988. PAF antagonists themselves had a profound effect, diminishing Ig production by the B-cell lines by up to 90%. These data indicate that PAF may have an important immunomodulatory role in B lymphocytes.

An ELISA spot assay for quantitation of human immunoglobulin-secreting cells.

The elucidation of changes in populations of immunoglobulin-secreting cells has been a cumbersome process. We present a simplified method for the enumeration of human immunoglobulin-secreting cells with an ELISA spot assay (ESA). This method is specific, will detect all isotypes, including IgE, and is sensitive, detecting as little as 10 pg of antibody secreted. In this article, we describe the methodology for performing the ESA, demonstrate the kinetics for optimal use in both B-lymphoblastoid lines and fresh B cells, and determine the correlation between the ESA and immunoglobulin secretion under various experimental conditions. This assay permits an estimate of the level of immunoglobulin secreted per cell, thus distinguishing expansion of immunoglobulin-secreting cell populations from increases in average (per cell) immunoglobulin production. The combination of ESA and quantitation of immunoglobulin in supernatants of cultured cells provides an easy and reliable means for studying the regulation of immunoglobulin secretion.

An open-label study of high-dose intravenous immunoglobulin in severe childhood asthma.

Eight pediatric patients with severe steroid-dependent asthma were enrolled in an open-label trial of high-dose intravenous immunoglobulin (IVIG) in an attempt to decrease their steroid requirements. Monthly therapy with high-dose IVIG resulted in a threefold decrease in both maintenance oral corticosteroid dose and in extra oral corticosteroids needed for control of exacerbations of asthma. This was accompanied by significant improvements in peak expiratory flow rates and in symptom-score rating. An immunomodulatory effect of IVIG was suggested by the changes in immediate skin test reactivity. Seven of the eight patients demonstrated one or more reactions to a panel of allergens before therapy. During the course of the trial, there was a progressive diminution in skin test reactivity with a 100-fold reduction in sensitivity at the completion of 6 months of therapy. In this preliminary study, the reduction in steroid requirements, improvement in symptoms and peak flow measurements, and diminution in immediate skin test reactivity support a potential role for IVIG in the treatment of severe steroid-dependent asthma. A larger, randomized trial now appears warranted.