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SUMMARY In this study, we have investigated the balance between Th1- and Th2-like activity in the lungs in sarcoidosis and have determined the effect of corticosteroid treatment on this. Twenty-one patients with acute untreated sarcoidosis were investigated by bronchoalveolar lavage (BAL) and compared with 11 normal volunteers. Sixteen of the sarcoid patients required corticosteroid therapy and seven of these were reinvestigated after 2–3 months' treatment. In order to assess Th1- and Th2-like activity in the lungs, IgG subclasses and IgE were measured in BAL fluid and serum, and IL-2, IL-4 and interferon-gamma (IFN-γ) in BAL. In patients with untreated sarcoidosis, albumin-corrected BAL/serum ratios for IgG4 and IgE were significantly reduced (IgG4, 1·04±0·18 (mean±s.e.m.); IgE 9·58±3·11) compared with those in normal controls (IgG4 5·3±0·72, P

ELISA is increasingly used to measure antibodies in new circumstances. Recently, it has been applied to the measurement of IgG subclass antibodies against common antigens in early childhood. These studies have raised concerns about the specificity of some of these assays. This paper details the results of experiments which have assessed the specificity of IgG 1 binding to allergens of dust mite (Dermatophagoides pteronyssinus) and ryegrass (Lolium perenne) pollen by inhibition ELISA in the sera of 2-year-old children of atopic parents. Six sera which showed binding of IgG 1 to D. pteronyssinus and six to L. perenne were used. All had IgG1 antibody against ovalbumin. In the children's sera, binding to D. pteronyssinus was substantially inhibited by preincubation with the homologous antigen, but not with ovalbumin, thereby confirming the specificity of the assay. However, suppression of IgGl binding to L. perenne with the homologous antigen was comparatively small, and ovalbumin could cause an equivalent inhibition, indicating poor specificity. Furthermore, the level of IgG1 binding to L. perenne was closely correlated to the level of IgG1 binding to ovalbumin (r= 0.98; P < 0.001). When the assay was reversed, IgG1 binding to ovalbumin was only slightly inhibited by L. perenne, indicating that most antibody binding to ovalbumin was specific. Thus, binding IgG1 in both adult and child sera to D. pteronyssinus appeared to be specific, while child, but not adult, IgG1 binding to L. perenne showed poor specificity. This disparity may be due to differences in the affinities of the respective antibodies, and it illustrates the importance of determining assay specificity when making measurements in early childhood.

Summary Previous work has suggested that there is a genetic predisposition for the development of both asthma and atopy. A recent study has also shown that there is a striking link between chromosome 11q and the IgE response underlying asthma and rhinitis. To further assess the linkage between chromosome 11q and atopy, we have studied nine families of two and, in many instances, three generations with the index case having asthma and/or atopy. Using two restriction fragment length polymorphism probes associated with the regions 11q12-q13.2, namely PYGM and INT2, we have been unable to confirm a significant link between this region of chromosome 11q and atopy as defined by a positive skin-prick test and or a raised specific IgE and/or a raised total IgE.

Summary A dog hair and dander (DHD) extract was prepared from hair obtained from mixed breeds. By SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting, using sera from 32 dog-allergic subjects, a number of IgE radio-staining bands could be seen. In 78% of sera a protein of molecular weight (MW) of 21 000 daltons, designated Ag X, was found to bind IgE and in 34% it did so strongly. This allergen was isolated from DHD by size-exclusion and ion exchange chromatography. The final product was a single allergen of MW of 21 000 and an isoelectric point of approximately 5.2. An additional protein-staining band could still be seen of MW of 24 000 daltons. Using a serum which contained IgE antibodies only to Ag X, this allergen was found only in DHD extract and dog saliva and was absent from dog serum and urine. It was the same dog allergen that we [1] reported as Ag 8 using crossed radio-immunoelectrophoresis (CRIE) and that Blands et al. [2] and Lowenstein [3] described as Ag 13. We propose that this major dog allergen be given the title Can f I according to the new allergen nomenclature.

Responses to the house dust mite during infancy may be important determinants of asthma in susceptible individuals. This study assessed early IgG subclass antibody responses to Dermatophagoides pteronyssinus in children of atopic parents. Sixteen atopic and 15 non-atopic children were selected from a birth cohort, and atopic status was established according to follow up over the first two years. IgG1 and IgG4 antibodies to D pteronyssinus were measured by enzyme linked immunosorbent assay at 7 days and 3, 6, 12, and 24 months. In all children D pteronyssinus IgG1 fell at 3 months (indicating maternal antibody loss), rose progressively to 12 months, and waned at 24 months. D pteronyssinus IgG4 was only detectable at 7 days. Children who were atopic by 2 years and therefore at greater risk of asthma, tended to have higher D pteronyssinus IgG1 at 6 and 12 months. These data suggest greater exposure or responsiveness to dust mite during infancy than in the second year.

This follow-up study of 191 babies investigated the development of food allergy in an unselected population and its relationship to total and antigen-specific IgE and IgG subclass levels. Sensitization to egg, as indicated by a positive skin test or RAST, was found in 5% of 1-year-old babies, but none of the babies in this series fulfilled the clinical criteria for immediate-type milk allergy. For both bovine casein (CAS) and egg albumin, the IgG response was largely restricted to IgG1 in contrast to the predominant IgG4 response to these antigens that is found in adults. The level of IgG4, but not IgG1, antibody to CAS and ovalbumin (OV) was lower in some of the babies compared with that of their mothers (N = 166; p less than 0.05, Student's paired t test). However, there was no difference in the total serum IgG subclass levels between mothers and babies. These results demonstrate that, in the population of babies studied, (1) type I hypersensitivity to egg occurred in 5% of 1-year-old babies, (2) the predominant IgG subclass of antibodies to CAS and OV in babies is IgG1, and (3) in the 22% of babies, there was substantially (greater than 1000-fold) less IgG4 antibody to CAS and OV than in their mothers, suggesting specific exclusion of some IgG4 antibodies.

Previous work has demonstrated a close association between certain histocompatibility antigens and the gene that controls the IgE response to certain ragweed allergens. For example, there is a 90% association between IgE production to the short ragweed allergen, Amb a V, and an HLA class II allele. To assess whether these HLA linkages are specific for ragweed, we have investigated the association between HLA antigens and the capacity of individuals to mount a specific IgE response to melittin in patients with bee-venom allergy. Twenty-two subjects with bee-venom sensitivity, 22 healthy beekeepers without bee-venom allergy, and a normal population of 149 unselected individuals were studied. With serologic tissue typing and restriction fragment length polymorphism analysis, we have demonstrated a significant decrease in the HLA-DR4 and DQw3 alleles in subjects who are allergic to melittin compared to the control populations. There was also a negative association between the presence of HLA-DR4 and DQw3 alleles with the capacity of the individuals to mount an IgE response to phospholipase A2 (PLA2). The bee-venom sensitive subjects had a slightly lower titer of anti-PLA2 IgG when these subjects were compared to the bee-venom insensitive beekeepers. These results support the view that either HLA-DR or HLA-DQ has a protective role in controlling the IgE immune response. Lack of an IgE response to melittin or PLA2 is unlikely to be due to a failure to recognize allergen.

The role of IgG-subclass antibodies in the spectrum of immunologic disorders has not yet been fully defined. In an attempt to understand its role in an immune complex-mediated disease, we studied patients who developed serum sickness (SSX) after treatment with an equine-derived immunoglobulin, antithymocyte globulin (ATG), for bone marrow failure. The predominant IgG subclass produced was IgGl, respresenting nearly 80% of all IgG anti-ATG activity present. The appearance of IgG anti-ATG antibodies and Clq-containing immune complexes was closely correlated with symptoms of SSX. Although other antibody isotypes were present and may have contributed to the patients' symptoms, it is evident that IgGl is the predominant IgG subclass produced in human SSX caused by a heterologous protein.

Since first proposed by Zamencik & Stephenson in 1978 [1], anti-sense oligodeoxynucleotides (ODN) have been successfully used to inhibit/block the expression of specific genes. This process relies on the ability of an ODN to bind a complementary sequence of messenger ribonucleic acid (mRNA) and so prevent its translation [2]. The mechanism is believed to operate through steric hindrance, by RNase H-like cleavage, or through a combination of both [3]. In addition, formation of triplex DNA via Hoogsteen or reverse-Hoogsteen binding may also have 'anti-gene' activity [4,5]. In the last few years there has been increasing interest in technology which allows researchers to address questions about gene function, to inhibit specific intracellular molecules and to temporarily block specific gene effects without the need for 'knock-out' mice or neutralizing antibodies [6]. This technology may lead to new therapeutic approaches to the treatment of human diseases at a molecular level, and trials are in progress to evaluate their potential for clinical use [7-9]. There are many advantages to anti-sense therapy [2,6,10]. Provided that the relevant gene sequences are known, there is very wide choice of gene targets, including intracellular, membrane-bound and soluble molecules of mammalian cells, bacteria and viruses. In this issue of Clinical and Experimental Immunology, Ikizawa et al. [11] describe the use of ODN to block IL-4 receptor expression and IgE synthesis by peripheral blood B cells. This editorial assesses ODN and their therapeutic potential.

The ability of CD8+ T cells to synthesise and secrete cytokines before and after culture with interleukin (IL)-4 and anti-interferon (IFN)-gamma was investigated. Purified rat splenic CD8+ and CD4+ T cells and CD4-CD8- cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for 6 h and expression of mRNA for IL-2, IL-4, IL-5, IL-6, IL-10 and IFN-gamma determined using a quantitative PCR technique. Secreted IFN-gamma and IL-2 were measured by ELISA and bioassay, respectively, 24 h after restimulation. The highest levels of IL-4, IL-5 and IFN-gamma mRNA were expressed by CD8+ T cells, the highest levels of message for IL-2 were expressed by CD4+ T cells and the highest levels of message for IL-6 and IL-10 by CD4-CD8- splenocytes. Purified rat splenic CD8+ T cells were cultured for 6 days with PMA, ionomycin and IL-2 with or without IL-4 or anti-IFN-gamma and restimulated with PMA and ionomycin. Those CD8+ T cells cultured with IL-4 expressed increased levels of IL-4, IL-5 and reduced levels of IL-2 and IL-6 mRNA, while CD8+ T cells cultured with IL-4 and anti-IFN-gamma expressed increased levels of mRNA for IL-10 and IFN-gamma. Comparable effects were seen for secreted IFN-gamma and IL-2. These results indicate that CD8+ T cells have the potential to produce large amounts of Th2 cytokines and are able to differentiate into different subpopulations with distinct cytokine profiles under the control of IL-4 and IFN-gamma.

Human primary effector T cells were analyzed for their susceptibility to anti-CD3-induced activation-induced cell death (AICD). Th1 and Tc1 cells were more susceptible to AICD than their type 2 counterparts. Type 1 and type 2 subsets were also found to be differentially susceptible to CD95-mediated apoptosis, although cell-surface expression of CD95 and CD95L was at similar levels on all subsets. A role for CD95 in AICD was confirmed by the addition of anti-CD95L antibodies that partially abrogated AICD. Residual apoptosis could not be accounted for by TNF-alpha/TNFR interactions because although type 1 cells secreted more TNF-alpha than type 2 cells, the addition of TNFR:Fc fusion protein did not inhibit AICD. Instead, a reduction in AICD was observed in the presence of EGTA or concanamycin A. The inhibition of apoptosis by a granzyme B inhibitor z-AAD-CMK in Tc1 cells further indicated an involvement of the granule exocytosis mechanism in AICD.

Differentiation of developing T cells into the type 1 (IFN-gamma-producing) or type 2 (IL-4-producing) subsets is a central theme of immune regulation. The balance of IL-4 and IL-12 present during T cell activation has been considered the major influence on type 1 versus type 2 development. Here we show that CD4 T cells can become biased towards type 1 or type 2 phenotypes during their initial activation in the absence of IL-4 or IL-12. This type of regulation is dependent on the balance of MAPkinase, protein kinase C, and calcineurin signaling after TCR engagement. Later maturation of Th1 or Th2 effectors is dependent on IL-12 or IL-4. However Tc1 CD8 effector development is independent of IL-12, and Tc2 cell generation requires both appropriate TCR signals and IL-4 early in effector development. Using an altered peptide ligand to stimulate TCR transgenic T cells, we show that altered signaling regulates the numbers of CD8 cells capable of developing into Tc2 effectors, and also their responsiveness to IL-4. Together, the results support a two-stage model of differentiation in which intermediate cells biased towards the type 1 or type 2 pathways after activation, are subsequently matured in response to IL-12 or IL-4, respectively.

The role of Immunoglobulin (Ig)E in inflammation is the subject of considerable study and a number of studies have shown conflicting evidence for its role in eosinophil recruitment and bronchial hyperresponsiveness in a number of murine models. The low affinity IgE receptor, CD23, is known to act as a negative regulator of IgE production and we have used knockout mice deficient in CD23 to investigate the role of IgE in eosinophil recruitment and bronchial hyperresponsiveness in a murine model of airway inflammation.To study the role of the low affinity FcepsilonII receptor, CD23 in IgE production, lung inflammation and bronchial hyperresponsiveness.Wild-type and CD23 knockout C57Bl/6 mice (CD23-/-) were immunized by intraperitoneal injection with ovalbumin on days 0 and 14 and challenged with aerosolized antigen on day 21 for a period of up to 1 week. Blood samples, bronchoalveolar lavage and lung tissue samples were obtained to determine serum IgE levels and inflammatory cell numbers, respectively. Furthermore, airway resistance was measured to increasing concentrations of aerosolized 5-hydroxytryptamine in order to evaluate the effect of CD23 deficiency on bronchial hyperresponsiveness to antigen challenge.Sensitization of wild-type C57Bl/6 mice to ovalbumin resulted in elevated levels of total serum IgE and ovalbumin-specific IgE, which was significantly augmented in CD23 knockout C57Bl/6 mice (CD23-/-). A significant increase in the percentage of eosinophils recovered in bronchoalveolar lavage fluid from wild-type and CD23-/- mice was observed 24 h following 3 or 7 days aerosol exposure with ovalbumin (10 mg/mL). At 3 days, the increase in the percentage of eosinophils was significantly greater in CD23-/- groups. Immunohistochemical analysis of lungs sections revealed the presence of CD3+, CD4+ and CD23+ cells in wild-type mice but a lack of immunofluorescence of CD23+ cells in CD23-/- mice. In wild-type ovalbumin-immunized mice, bronchial hyperresponsiveness to aerosolized 5-hydroxytryptamine was observed following a 3-day antigen challenge, which was significantly greater in CD23-/- ovalbumin-immunized mice.These studies demonstrate that CD23-/- mice have increased capacity to produce IgE consistent with the view of a negative feedback role for membrane-bound CD23 and under such conditions, may account for the greater numbers of eosinophils recruited to the airways and bronchial hyperresponsiveness observed following acute but not chronic antigen challenge.

The role of the cytokines interleukin-4 and interferon-γ in the regulation of IgE responses in the mouse and man have focused on the role of CD4 T cells. In the rat, antigen-specific CD8 T cells, generated following inhalation of antigen, have been shown to be capable of suppressing IgE responses. Repeated intraperi-toneal injections of 1 ng ricin and 1 μg antigen established a long-lived IgE response in both low- and high-IgE responder rat strains (Wistar and Brown Norway). The duration of the IgE antibody response was 204 and 248 days, respectively. Total IgE levels rose from 30 ± 20 to 39,000 ± 7,500 ng/ml in the Wistar rat and from 120 ± 100 to 47,000 ± 8,000 ng/ml in the Brown Norway rat. An even greater (104-fold) increase was seen in antigen-specific IgE antibody levels. Ricin alone had no effect and concomitant or prior stimulation with antigen was required. The proportion of CD4+ and CD8+ cells present in the spleen at the peak of the IgE response was markedly increased compared with animals given ricin or antigen alone. Furthermore, CD8 T cells were approximately 100 times more sensitive to ricin than CD4 T cells. These data suggest that enhancement of IgE responses in ricin-treated animals results from the selective deletion of T cells which suppress IgE and are of the CD8 phenotype.

Interferon-gamma (IFN-gamma) is an important cytokine involved in the regulation of allergen-induced immune responses. We examined the role of IFN-gamma in a Brown-Norway rat model of bronchial hyperresponsiveness (BHR) and airway eosinophilia, and its effects on the mRNA expression of T helper type 1 (Th1)/Th2 cytokine. Ovalbumin (OA)-sensitized animals were given either exogenous IFN-gamma (105 U/rat over 3 days, intraperitoneally) or anti-IFN-gamma blocking antibody (DB-1 0.3 mg/rat, intravenously) prior to exposure to OA aerosol and were studied 18-24 hr later. In sensitized animals, OA induced significant BHR, accumulation of eosinophils, T lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid, and also increased eosinophils and CD8+ T cells in the airways. Exogenous IFN-gamma attenuated allergen-induced BHR (P0.02, compared with sham-treated animals) together with a significant reduction in eosinophil and neutrophil numbers in BAL fluid (P0. 005), and eosinophils and CD8+ T cells in airways (P0.05). By contrast, anti-IFN-gamma antibody increased airway CD4+ T cells and BHR. Using reverse transcriptase-polymerase chain reaction, significant increases in Th2 [interleukin-4 (IL-4), IL-5 and IL-10], and IFN-gamma cytokine mRNA were found in the lungs of sensitized and OA-exposed animals, while exogenous IFN-gamma significantly suppressed IL-4, IL-5 and IL-10 mRNA expression, and anti-IFN-gamma antibody increased IL-4 and IL-5 mRNA expression. These results indicate that Th1 effects, such as those mediated by IFN-gamma, play a down-regulatory role to suppress the Th2 responses associated with allergen-induced BHR and eosinophilic inflammation.

In the following study, we demonstrate that medium responder PVG rats immunized i.p. with OVA complexed to the adjuvant aluminum hydroxide exhibit a moderate IgE response (400-1000 ng/ml). In these rats, we demonstrate that underlying the MHC class II-restricted CD4+ T cell response, there is an MHC class I-restricted CD8+ T cell component that plays an important role in restricting the magnitude and duration of the IgE response. We show that in vivo depletion of CD8+ T cells effects a massive increase in IgE (20-fold), and that they are MHC class I-restricted, OVA-specific, cytolytic cells that universally produce IFN-gamma (25-69 ng/ml) and IL-2 (7.6-22 U/ml), and occasionally secrete IL-4 (68-81 U/ml IL-4), and when adoptively transferred into CD8-depleted recipients, can effect a significant reduction in IgE (3- to 50-fold). We also demonstrate that this in vivo inhibition of IgE is dependent on the Ag-specific activation of the CD8+ T cells, and that the activated CD8+ T cells will suppress total/bystander IgE in an Ag-nonspecific manner. These data are consistent with a growing literature demonstrating sensitization of MHC class I-restricted CD8+ T cells by exogenous protein Ags delivered to mucosal sites, and may represent a mechanism whereby a selective pressure can be applied on the functional outcome of an immunoglobulin response to environmental allergens.

Previous studies have demonstrated an infiltration of monocytes and increased levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the asthmatic lung. To study the possible effects of this cytokine upon the differentiation and function of these newly recruited monocytes, we have developed a model in which monocytes isolated from human peripheral blood were differentiated into macrophages in serum in the presence or absence of GM-CSF. After 7 days, the macrophages increased in size and granularity, had increased phagocytic activity, and expressed various adhesion molecules, CD14 and major histocompatibility complex (MHC) class II. The effects of GM-CSF on antigen presentation by cultured macrophages on the antigen-specific proliferative response of CD4+ T cells to Dermatophagoides pteronyssinus or purified protein derivative of tuberculin and the mitogen phytohaemagglutinin was determined. CD4+ T-cell proliferation was reduced when either antigen was presented by macrophages cultured in serum alone, compared with the values obtained with freshly isolated monocytes. However, CD4+ cell proliferation was comparable to that observed with monocytes when antigen was presented by macrophages which had been pre-cultured with 50 U/ml GM-CSF. CD4+ T-cell proliferation to phytohaemagglutinin was similar when all three populations were used as accessory cells. High numbers of macrophages partially suppressed CD4+ T-cell proliferation in response to antigen presented by monocytes, but there was no significant difference between macrophages cultured in the presence or absence of GM-CSF. This data suggests that GM-CSF directs monocyte differentiation into macrophages with an antigen-presenting, rather than a suppressive, phenotype. Elevated levels of GM-CSF in the asthmatic lung may therefore maintain recently recruited monocytes in an inflammatory and T-cell activating state.

Following allergen exposure, sensitized Brown-Norway rats develop airway hyperresponsiveness (AHR) and eosinophilic inflammation together with an increase in activated T cells (CD25+) in the airways. We tested the hypothesis that CD4+ T cells are involved directly in the acquisition of AHR. Spleen T cells from animals that were injected intraperitoneally on three consecutive days with ovalbumin/Al(OH)3, showed a dose-dependent proliferative response in vitro to ovalbumin, but not to bovine serum albumin, as measured by [3H]thymidine uptake. For total T-cell transfer, spleen cells obtained from donor rats 4 days after sensitization were depleted of adherent cells by a nylon wool column separation. CD4+ and CD8+ T cells were purified by immunomagnetic beads cell separation. Recipient naive rats were injected intravenously with 50 x 10(6) total T cells, 20 x 10(6) and 5 x 10(6) CD4+ cells, and 5 x 10(6) CD8+ cells, and were exposed to ovalbumin aerosol 24 hr afterwards. After a further 24 hr, airway responsiveness to acetylcholine (ACh) was measured and provocative concentration (PC) values PC100, PC200 and PC300) (the ACh concentration needed to achieve 100, 200 and 300% increase in lung resistance above baseline) were calculated. Airway responsiveness was significantly increased in recipients of sensitized total T cells compared with recipients of cells from saline-injected donor rats (P < 0.05). There were significantly increased eosinophil major basic protein (MBP)+ cell counts/mm2 in airway submucosal tissue in the hyperreactive rats and a significant correlation was found between the number of MBP+ cells and PC100 (r = 0.75; P < 0.03) in recipients of sensitized total T cells. Purified CD4+ T cells from sensitized donors induced AHR in naive recipients (P < 0.05), while sensitized CD8+ and naive CD4+ cells failed to do so. Our data indicate that T cells may induce AHR through an eosinophilic airway inflammation and that CD4+ T cells may have a direct effect in this process in Brown-Norway rats.

The role of CD8+ T cells in the regulation of IgE was investigated by in vivo CD8+ T-cell depletion. Following intraperitoneal immunization with ovalbumin (OVA), OVA-specific T cell responses were first detected in the draining lymph nodes (LN) and subsequently in the spleen. In vitro depletion of LN CD8+ cells reduced the OVA-specific LN CD4+ T cell response; while depletion of splenic CD8+ cells enhanced the OVA-specific splenic CD4+ T cell response, suggesting an early helper and later suppressor function. CD8+ cell depletion in vivo up to 7 days after immunization failed to enhance IgE production, but depletion of CD8+ T cells between day 12 and day 18 increased IgE levels to over 10 microg/ml. Adoptive transfer of 1 x 10(7) purified CD8+ T cells (95% CD8+98% CD3+1% NK,1% CD4+) from parallel immunized rats or of 1 x 106 cells of an OVA-specific, MHC class I restricted CD8+ T cell line suppressed the IgE, but not IgG, response by95%. These results provide further evidence that CD8+ T cells inhibit IgE production, possibly by preventing the generation of adequate amounts of appropriate CD4+ T cell help.

Chemical allergens of different types, those that cause in humans allergic contact dermatitis or occupational asthma induce in mice divergent immune responses characteristic, respectively, of T-helper 1 (Th1)- and Th2-type cell activation. Such responses are associated with the development of different cytokine secretion patterns by draining lymph node cells (LNC), such that contact allergens stimulate vigorous interferon-gamma (IFN-gamma) production, but little secretion of the Th2 cytokines interleukin-4 and interleukin-10 (IL-4 and IL-10), whereas the converse pattern is provoked by respiratory allergens. Using selective depletion with antibody and complement we have here examined the relative contribution of CD4+ and CD8+ T lymphocytes to the cytokine secretion patterns of draining LNC isolated from mice sensitized to chemical allergens. Mice received repeated topical applications of respiratory allergens, trimellitic anhydride (TMA) or diphenylmethane diisocyanate (MDI), or of contact allergens 2,4-dinitrochlorobenzene (DNCB) or formaldehyde. Thirteen days following the initiation of exposure the production by draining LNC of IL-10, IFN-gamma and mitogen (concanavalin A)-inducible IL-4 was measured by enzyme-linked immunosorbent assay (ELISA) after various periods of culture. It was found that the high levels of IL-4 and IL-10 secretion stimulated by TMA or MDI, and the lower levels of these cytokines induced by DNCB or formaldehyde, were in all cases dependent upon the presence of CD4- cells. In contrast, the comparatively high concentrations of IFN-gamma observed following exposure to contact allergens were found to be derived from CD4+ cells, and in the case of DNCB from CD8+ cells also. The low levels of IFN-gamma induced by treatment with TMA or MDI were associated largely or wholly with CD8+ cells. These data indicate that the type 2 cytokine responses induced to different extents by both contact and respiratory chemical allergens are almost exclusively a function of CD4+ cells, but that IFN-gamma is produced by either CD4+ cells in the case of contact allergens or largely by CD8+ cells in the case of chemical respiratory allergens.

We determined the expression of Th-2 type cytokines, interleukin-4 (IL-4) and IL-5, and of the Th-1 type cytokine, interferon-gamma (IFN-gamma), in the Brown-Norway rat. Rats were intraperitoneally sensitized with ovalbumin and 21 days later were either exposed to ovalbumin or saline aerosol. The value -log PC300 (PC300 = concentration of acetylcholine needed to increase baseline lung resistance by 300%) was 2.49 +/- 0.15 in sensitized, exposed rats, was higher than in sensitized, saline-exposed or naive rats (1.54 +/- 0.27 and 1.63 +/- 0.06 respectively, P < 0.05). There was a significant increase in eosinophils in bronchoalveolar lavage fluid and in airway submucosal airway tissues in the sensitized exposed group. Reverse-transcriptase polymerase chain reaction was performed on total lung RNA using primers for IL-4, IL-5, IFN-gamma and beta-actin. IL-4 and IL-5 mRNA levels in control and sensitized saline-exposed rats were not detectable, but increased levels were found in sensitized and ovalbumin-exposed rats with levels of 0.25 +/- 0.01 and 0.98 +/- 0.02% of beta-actin mRNA as assessed by densitometric measurements. Expression of IFN-gamma mRNA was significantly reduced in sensitized and ovalbumin-exposed rats. As in asthmatic airways, there is an increased expression of Th-2 cytokines, IL-4 and IL-5, together with a reduction in the Th-1 cytokine, IFN-gamma, thus supporting a role for Th-2 cytokines in allergic eosinophilic inflammation.

In this study, we have investigated the effects of IL-4 and IFN-gamma on the growth and differentiation of CD8+ T cells in vitro. Rat splenic CD8+ T cells expressed higher levels of IL-4, IL-5, IL-10, and IFN-gamma mRNA, as measured by reverse-transcription PCR, than CD4+ T cells from the same source, whereas CD4+ T cells expressed more IL-2 and IL-6 mRNA. CD8+ T cells were cultured for 6 days with PMA, ionomycin, and IL-2, and their ability to proliferate, to express mRNA for IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-gamma, and to secrete IFN-gamma and IL-2 was determined. IL-4 could act as a growth factor for CD8+ T cells during primary stimulation, but inhibited proliferation of the restimulated 6-day-cultured CD8+ T cells. The highest levels of mRNA for IL-2 and IL-6 were detected in control cultures in which little mRNA for IL-4, IL-5, or IL-10 was detected. Addition of IL-4 to the primary cultures reduced the capacity of the restimulated cells to express mRNA for IL-2, and for IL-6, but enhanced expression of mRNA for IL-4 and IL-5. Addition of IFN-gamma to the cultures, alone or in conjunction with IL-4, or addition of IFN-gamma-specific neutralizing Ab, had little effect. However, in conjunction with IL-4, anti-IFN-gamma enhanced expression of mRNA for IFN-gamma, and for IL-10. These results indicate that IL-4 and IFN-gamma regulate the differentiation of CD8+ T cells and the growth of cytotoxic and other types of CD8+ T cells, and suggest pathways through which CD8+ T cells may interact with other immune cells.

Interleukin-4 (IL-4) is antagonistic for many of the activities of interferon-gamma (IFN-gamma) and, as well as suppressing the development of T-helper type-1 (Th1) cells, has been reported to block directly the synthesis of IFN-gamma in human lymphocytes. However, IL-4 transgenic mice produce increased amounts of IFN-gamma as well as IL-4. We have compared the ability of rat IL-4 to regulate IFN-gamma secretion in short-term cultures of spleen cells with its effect on the differentiation of T lymphocytes into IFN-gamma-producing, or Th1-type, cells. Normal rat spleen cells were stimulated using a variety of mitogens and ovalbumin antigen, with or without IL-4, for 12-24 hr and the levels of IFN-gamma in the supernatants measured by enzyme-linked immunosorbent assay (ELISA). The results show that when normal rat splenocytes were stimulated with phytohaemagglutinin (PHA) or concavalin A (Con A), IL-4 enhanced secretion of IFN-gamma after 12-24 hr. This enhancement was also apparent when splenocytes from animals immunized 10 days previously with alum-precipitated ovalbumin were stimulated with ovalbumin in vitro, and appeared to be mediated primarily via CD+ T cells. In contrast, when spleen cells were maximally stimulated with phorbol myristate acetate (PMA) and ionomycin, addition of IL-4 had no effect on the amount of IFN-gamma secreted. When splenocytes were stimulated with Con A for 4 days in the presence of IL-4, and restimulated with PMA and ionomycin, IFN-gamma secretion was greatly suppressed. Our results indicate that IL-4 exerts differential effects on IFN-gamma secretion and on the development of IFN-gamma-producing lymphocytes.

Ricin has been shown to enhance IgE production in the rat, probably through inhibition of suppressor T lymphocytes. We have studied further the effects of ricin on IgE titre and have determined its effects on antigen-induced airway inflammation and hyperreactivity in the Brown Norway rat. Immunization with ovalbumin (1-100 micrograms, intraperitoneally) produced dose-related increases in serum antigen-specific IgE titre. Ricin augmented the total IgE titre and caused about a 10-fold increase in the peak antigen-specific IgE titre. In sensitized animals, antigen challenge (three times with aerosolized ovalbumin every second day) caused a significant influx of eosinophils and neutrophils and significant airway hyperreactivity 24 hr after the third challenge. In sensitized animals that had also received ricin, the eosinophil and neutrophil influx was further significantly potentiated and a significant influx of lymphocytes also occurred. Thus, there was a relationship between the degree of sensitization and the magnitude of the inflammatory response. However, the enhanced airway inflammation in ricin-treated animals was not accompanied by a further enhancement of airway hyperreactivity. The present study demonstrates that ricin enhances IgE production and augments an antigen-induced inflammatory pathology but does not potentiate antigen-induced airway hyperreactivity.

The ability of CD8+ T cells to synthesise and secrete cytokines before and after culture with interleukin (IL)-4 and anti-interferon (IFN)-gamma was investigated. Purified rat splenic CD8+ and CD4+ T cells and CD4-CD8- cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for 6 h and expression of mRNA for IL-2, IL-4, IL-5, IL-6, IL-10 and IFN-gamma determined using a quantitative PCR technique. Secreted IFN-gamma and IL-2 were measured by ELISA and bioassay, respectively, 24 h after restimulation. The highest levels of IL-4, IL-5 and IFN-gamma mRNA were expressed by CD8+ T cells, the highest levels of message for IL-2 were expressed by CD4+ T cells and the highest levels of message for IL-6 and IL-10 by CD4-CD8- splenocytes. Purified rat splenic CD8+ T cells were cultured for 6 days with PMA, ionomycin and IL-2 with or without IL-4 or anti-IFN-gamma and restimulated with PMA and ionomycin. Those CD8+ T cells cultured with IL-4 expressed increased levels of IL-4, IL-5 and reduced levels of IL-2 and IL-6 mRNA, while CD8+ T cells cultured with IL-4 and anti-IFN-gamma expressed increased levels of mRNA for IL-10 and IFN-gamma. Comparable effects were seen for secreted IFN-gamma and IL-2. These results indicate that CD8+ T cells have the potential to produce large amounts of Th2 cytokines and are able to differentiate into different subpopulations with distinct cytokine profiles under the control of IL-4 and IFN-gamma.

We have systematically investigated the capacity of highly purified human peripheral CD8+ T cells to produce interleukin (IL)-4 and interferon (IFN)-gamma when triggered by different stimuli. CD8+ T cells were isolated from peripheral blood by positive selection to99% purity and stimulated with one of three different stimuli: phytohaemagglutinin (PHA) and IL-2, phorbol myristate acetate (PMA) and ionomycin, and plate-bound anti CD3 and PMA. On their own, ionomycin and IL-2 failed to stimulate significant CD8+ T cell proliferation while PHA, plate-bound anti-CD3 and PMA induced weak proliferation. A combination of PHA and IL-2, PMA and ionomycin, or plate-bound anti-CD3 and PMA all induced vigorous CD8+ T cell proliferation. IFN-gamma was produced following all three stimuli, but was greatest from cells cultured with PMA and ionomycin. However, IL-4 secretion was only detected in cell cultures stimulated with PMA and ionomycin. These results indicate that, with sufficient stimulation, human CD8+ T cells have the potential to produce Th2 as well as Th1 cytokines.

The effect of ricin treatment on IgE levels and airway inflammation has been examined in ovalbumin (OA)-sensitized rats. Ricin augmented the total IgE titre and caused about a 10-fold increase in the antigen-specific IgE titre. Repeat antigen challenges (every second day) with an OA aerosol increased the number of eosinophils and neutrophils in bronchoalveolar lavage fluid and produced airway hyperresponsiveness (AHR) 24 h after the third challenge. Ricin pretreatment caused a significantly larger influx of eosinophils, neutrophils and lymphocytes into lavage fluid, but there was no further increase in AHR. The present study demonstrates that ricin augments IgE production and the inflammatory response, but that AHR is unrelated to the number of infiltrating inflammatory cells.

The link between immunoglobulin E (IgE) antibodies and environmental allergens, allergic sensitization and atopic diseases such as asthma, rhinitis and eczema is well established, yet treatment of these diseases is largely symptomatic and fails to correct the underlying immune disorder. B-cell production of IgE depends on interleukin 4 (IL-4) and is inhibited by interferon (IFN-γ). These cytokines are produced by T-helper 2 (Th2) and T-helper 1 (Th1) CD4+ T cells respectively. In atopic dermatitis skin and asthmatic lung, Th2 cells predominate but antigen-specific CD4+ T-cell clones derived from peripheral blood are a mixture of Th1, Th2, and Th0. Circulating allergen-specific CD4+ T cells differentiate into Th1- and Th2-like clones in the presence of IL-12 and IL-4 respectively. In rats, CD8+ T cells are potent regulators of IgE responses in vivo, although the mechanism for this is still unclear. CD8+ T-cell depletion in immunized animals prolonged the IgE response but prior depletion did not. Indeed, prior depletion of CD8+ T cells actually inhibited IgE production, suggesting that there may be more than one type of IgE regulatory CD8+ T cell. This was supported by the observation that CD8+ T cells inhibited the differentiation of both Th1 and Th2 CD4+ T cells in vitro as well as IL-4-but not IL-2-induced Th2 CD4+ T-cell growth. The existence of distinct CD8+ T-cell subsets is supported by the observation that IL-4 and IFN-γ regulate the growth and differentiation of CD8+ T cells. IL-4 inhibits the development of CD8+ T cells that produce IL-2 and IL-6 and enhances the generation of cells that make IL-4 and IL-5. A combination of IL-4 and anti-IFN-γ enhanced IFN-γ and IL-10 production. Studies are continuing to characterize different CD8+ T-cell subsets at a clonal level and to determine their role in IgE regulation.

Immunization of rats with soluble antigen (ovalbumin) and the castor bean toxin, ricin, eliminates a subpopulation of CD8+ T cells which suppress IgE responses in vivo. This treatment also reduces the ability of splenic T cells to produce interferon-gamma (IFN-gamma) and enhances their capacity to make interleukin-4 (IL-4). In this report we describe the effect of immunization with ricin and antigen on the expression of mRNA for other T-helper type 2 (Th2) cytokines--IL-5 and IL-10--and their relationship to serum IgE and IL-4 mRNA expression. Splenocytes were taken from rats at different times after immunization with antigen or ricin and antigen and activated in vitro with phorbol myristate acetate (PMA) and ionomycin for 6 hr and total RNA extracted and reverse transcribed. Cytokine gene expression was detected using a quantitative polymerase chain reaction (PCR). Expression of IL-4, IL-5, and IL-10 was increased 7-20-fold 11 days after immunization with ricin and antigen (from 0.107% to 0.769% beta-actin for IL-4, from 0.0167% to 0.381% beta-actin for IL-5, and from 0.0581% to 0.954% beta-actin for IL-10), and preceded maximum serum IgE levels by 4-5 days. There was no increase in IgE or mRNA for these cytokines in rats immunized with antigen alone. The level of IL-4, IL-5, and IL-10 expression declined rapidly after 12 days. Our results suggest that immunization with antigen and ricin preferentially induces a Th2 response, and that CD8+ T cells may play a part in down-regulating the development of Th2 T cells.

Ricin, a toxic lectin from castor beans greatly enhances IgE responses to bee venom phospholipase A2 (PLA2) in high and low IgE responder strains of rat. The increase in IgE is accompanied by a 60% reduction in the number of CD8+ but not CD4+ T cells in the spleen. Optimal enhancement of IgE by ricin occurs when it is given at the same time as the antigen or 24 hr later, suggesting that it acts on cells which were activated as a consequence of immunization. Radio ligand-binding studies with 125I ricin were used to compare the number of ricin binding sites on CD4+ and CD8+ T cells. No difference was seen in either the affinity or the number of receptors for ricin on the CD4+ and CD8+ T cells of unimmunized rats. In contrast, CD8+ T cells taken from rats which had been immunized with 10 micrograms of PLA2 24 hr earlier demonstrated considerably more ricin receptors (3.9 x 10(7) +/- 2.2 x 10(6) binding sites/cell) than CD4+ T cells (3.19 x 10(6) +/- 1.08 x 10(6) binding sites/cell). However the affinity of the receptors for ricin was unchanged. Cytofluorographic analysis with fluorescein isothiocyanate (FITC)-labelled ricin confirmed these observations and indicated that increased ricin binding occurred on a subpopulation of CD8+ T cells. The effect of CD8+ T cells on IgE regulation was investigated by adoptive transfer. 1 x 10(8) highly purified (> 98%) splenic CD8+ T cells collected from Brown Norway rats 3 days after immunization with 10 micrograms of PLA2 were adoptively transferred to naive, syngeneic recipients. The IgE antibody response to PLA2 + A1(OH)3 seen in these animals was reduced by 91%. Adoptive transfer of CD4+ T cells from the same donor animals did not induce suppression and nor did adoptive transfer of CD8+ T cells from animals given both ricin and PLA2. However, when recipients of CD8+ T cells taken from rats immunized with PLA2 were immunized with a different antigen [ovalbumin (OVA)] and A1(OH)3 the IgE antibody response was also suppressed, although to a lesser extent (66%). These results show that co-administration of ricin and PLA2 depletes a subpopulation of ricin-sensitive, early activated CD8+ T cells and that these CD8+ T cells are potent suppressors of the primary IgE response.

Previous studies have suggested that there is a genetic predisposition for the development of asthma and atopy. A recent study has also demonstrated that there is a striking link between chromosome 11q and the IgE response underlying asthma and rhinitis. To assess the linkage between chromosome 11q (region D11S97) and atopy or bronchial hyperresponsiveness (BH), we have studied nine families of two and, in many instances, three generations with the index case having asthma and/or atopy. With variable number of tandem repeat analysis with the probe, p lambda-MS.51, we have been unable to confirm a significant link between region D11S97 of chromosome 11q and either atopy or BH to methacholine. We have demonstrated that atopy and BH produce similar log of odds scores with linkage analysis at each recombination fraction from 0.001 to 0.5 with both Hinf1 and Taq1 restriction digests and that the use of either a positive skin prick test or positive RAST as a definition of atopy does not significantly alter the log of odds score.

Summary This study investigates the early humoral immune response following natural exposure to an inhalant antigen (Dermatophagoides pteronyssinus) in 36 babies, from birth until 1 year of age. The total IgG and subclass 1 and 4 D. pteronyssinus-specific antibody levels were assayed in sera collected at 7 days, 3 and 12 months by ELISA. After an initial fall, due to the progressive loss of maternal antibodies, an IgG specific response to D. pteronyssinus was seen between 3 and 12 months. This was restricted to the IgG1 subclass when the values at 12 months were significantly higher than those detected at the third month (P < 0.001, paired t test). No D. pteronyssinus-specific IgG4 antibody was detected in any subject at any of the time points tested. The present study demonstrates that inhalant as well as food antigens are able to stimulate the immune system during the first year of life and that the antibodies produced are of the IgG1 subclass.

Antibody activity in the major classes and IgG subclasses against antigens in factory humidifier water was quantified by enzyme immunoassay (EIA) in 88 subjects who were exposed at work to the output from these contaminated humidifiers. Those with work-related symptoms had significantly higher mean titres than those who were symptom free, although values overlapped. The individuals with the highest IgG antibody titres also had the highest titres of IgM and IgA antibody, and these parameters did not discriminate between those with and without symptoms any better than the IgG titre. This was also true for the IgG subclasses where activity was predominantly measured in IgG1. Quantifying the IgG antibody allowed us to demonstrate a significant correlation with years of work exposure (P less than 0.001). There was no significant association between antibody and cigarette smoking, as assessed by smoking history and confirmed objectively by serum cotinine levels. There was a significant correlation with total IgG level (P less than 0.001) suggesting that a non-specific immune enhancement may accompany the specific response. The antibody titres were followed up to 3 years after modification of the humidification systems, and during this time symptoms resolved and the antibody levels progressively fell to undetectable levels. The EIA was adapted to measure antigen at nanogram levels thus providing a rapid test for screening of humidifer water as well as a technique that may help identify the nature of the antigens involved.

Screening for atopic disease using neonatal serum IgE has been advocated on the basis of the predictive value of elevated levels. However, this is only one measure of validity. The test was validated fully in 92 infants with a bi-parental history of atopy using 0.7 IU/ml as the cut-off. All infants were assessed prospectively for evidence of atopic disease (eczema, recurrent wheezing or food reactions) and skin-prick test positivity in the first year. Total serum IgE was measured by ultrasensitive ELISA on 61 cord blood samples and 92 samples taken at 7 days. All cord samples were re-analysed by PRIST and the first 33 by ultrasensitive RIA giving, respectively, 82% and 94% concordance (regarding undetectable, detectable and elevated levels) with ELISA. Maternal contamination was indicated in 7% of cord samples by high serum IgA. Ninety-five per cent of cord/7-day IgE pairs showed no change or minor rises at 7 days. Forty-nine per cent of the infants had evidence of atopic disease. Only 5% had elevated 7-day IgE. The positive and negative predictive values of the 7-day test were 60% and 52%, respectively, and specificity 96% but the sensitivity was only 7%. High levels did not distinguish the infants with the most unequivocal evidence of disease, i.e. eczema with a positive skin test. In conclusion IgE at 7 days is comparable to and more reliable than cord IgE. However, neonatal IgE screening is too insensitive to have clinical application.

Certain strains of rats infested with the nematode parasite Nippostrongulus brasiliensis mount vigorous, persistent immunoglobulin E (IgE) responses. In the absence of parasites, adjuvants such as Bordatella pertussis or Al(OH)3 are needed to produce IgE responses to soluble antigens. These are short-lived, even in high IgE responder strains. In this study we have produced long-lived IgE responses in both low (Wistar) and high (Brown Norway) IgE responder strains of rats by repeated injections of ricin, a toxic lectin from castor beans, and phospholipase A2 (PLA2), a bee venom protein. Total IgE levels rose from 30 +/- 20 ng/ml to 39,000 +/- 7500 ng/ml in the Wistar rats compared with an increase from 120 +/- 100 ng/ml to 47,000 +/- 8000 ng/ml in the Brown Norway rats. An even greater (10(4)-fold) increase was seen in PLA2-specific IgE antibody levels. total and PLA2-specific IgE started to fall 6 weeks after treatment was stopped in the Wistar and after 12 weeks in the Brown Norway rats. The duration of the response was 204 and 248 days, respectively. The IgE-enhancing properties of ricin were compared in low, mid (Hooded Lister) and high IgE responder rats. Total IgE and PLA2-specific IgE but not IgG antibody (Ab) responses were enhanced in all animals given ricin and PLA2 but not in animals given ricin or PLA2 alone. The increase was greater in Wistar rats (48-fold) than in Brown Norway rats (eightfold) and by Day 24 the levels of both total and PLA2-specific IgE in three different strains were indistinguishable. PLA2-specific IgE antibody-secreting cells were detected in the spleen at a frequency of 1:5000. These results show: (i) that repeated immunization of rats with antigen and ricin produce a very large IgE response which was long-lived; (ii) that this response was indistinguishable in different IgE responder strains of rat; and (iii) that the IgE response declines earlier in low IgE responder strains of rats.

Since the discovery ofIgE in the late 1960s (Ishizaka, Ishizaka & Hornbrook, 1966; Johansson & Bennich, 1967) several animal models have been developed to study the regulation of IgE. IgE responses in atopic people are typically long lived and can be maintained when they are no longer exposed to allergen as in hay fever patients outside the pollen season. Such responses have proved difficult to produce in most species, with the exception of mice, and after an initial burst of IgE when first immunized, the response generated is rapidly suppressed. The artificial induction of IgE responses in rodents has required the use of adjuvants such as aluminium hydroxide and Bordatella pertussis or concomitant nematode infection (Jarrett & Stewart, 1972). T cells play a central role in regulating IgE responses (Okumura & Tada, 197 la; Katona, Urban & Finkelman, 1988), and as well as providing help for IgE B cells, they are capable of suppressing both primary and secondary IgE responses (Okumura & Tada, 1971b; Holt, Batty & Turner, 1981a). Thus two opposing functions for T cells in IgE regulation can be seen. These effects are mediated at least in part by T cell-derived cytokines. High levels of interleukin-4 (IL-4) were found to potentiate IgE synthesis from lipopolysaccharide (LPS) stimulated murine B cells in vitro (Coffman & Carty, 1986) and this effect could be antagonized with interferon-gamma (IFN-y) (Snapper & Paul, 1987). Anti-IL-4 antibody (Finkelman et al., 1986, 1988a) or IFN-y (Finkelman et al., 1988b) both suppress antigen and Nippostrongylus braziliensis-induced IgE responses in vivo. With N. braziliensis infestation, IgE levels spontaneously decline following worm expulsion but similar effects were also seen with anti-IL-4 and IFN-y in Heligmosomoides polygyrusinfested mice, where the worm is not expelled and IgE levels do not fall (Finkelman et al., 1990). The secondary IgE response produced with antigen and Al(OH)3 was largely but not completely inhibited with anti-IL-4 (Finkelman et al., 1990); these investigators conclude that the role of IL-4 in the maintenance of an IgE response is to recruit

A double-blind, placebo-controlled trial of immunotherapy was conducted in patients with Dermatophagoides pteronyssinus rhinitis. Thirty patients received an extract with a high content of Der p I (Pharmalgen), 20 received a conventional mite extract (Allpyral), and 30 patients received histamine chloride (placebo). Specific IgG and subclasses were measured before and after 3 and 12 months of treatment by RIA and/or ELISA, and specific IgE by RAST. Clinical outcome was assessed by skin prick tests, nasal challenge, visual analogue, and diary-card symptom and drug scores; from these findings, a clinical index was derived. An IgG response occurred only in the Pharmalgen-treated group: D. pter IgG and IgG1 increased by 3 months (p less than 0.05) and then plateaued to 12 months (p less than 0.05). IgG4 levels increased throughout treatment (p less than 0.05 and p less than 0.01), as did the IgG/IgE ratio. A subclass switch from IgG1 to IgG4 occurred. D. pter IgE rose at 3 months (p less than 0.05). Clinical improvement occurred at 3 and 12 months in the Pharmalgen-treated group only. Pretreatment levels of IgE, IgG1, or IgG4 did not predict clinical outcome. Our findings are compatible with the hypothesis that IgG subclasses may modulate antigen-IgE interactions, although the antibody response to this potent extract need not be causally related to improvement.

Summary During immunotherapy with wasp venom, levels of venom-specific IgE antibodies increase and then fall, whereas the concentration of IgG antibodies rises and then remains at a high level. Successful treatment is therefore associated with both increased concentrations of serum IgG and decreased serum IgE antibodies to venom. In this study we have investigated the possible role of auto-antibodies in inducing the decrease in serum IgE antibody. Levels of auto-anti-IgE were measured by a radioimmunoassay. Anti-IgE auto-antibodies were not generated during immunotherapy and there was no significant difference in the levels of anti-IgE auto-antibodies between patients whose venom-specific IgE antibody levels fell more than fivefold after immunotherapy and those patients in whom IgE antibody levels did not change significantly. We conclude that anti-IgE auto-antibodies do not play a part in IgE suppression induced by immunotherapy.

461 Distribution o f Plasma Cell Markers and Intracellular IgE in t h e Cell L i n e U266. E Lagging BS, M van HageHamsten MD. B H~rfast PhD, G Halld~n PhD, Stockholm, SWEDEN We made use of the IgE producing plasma cell line U266 in order to be able to stud)' intracellular IgE and the distribution of the plasma cell markers CD38 and PCA-I. The cell were penneabilized according to the FOG method (G Hallddn, J lmmunol Methods 124. 1989) and analysed with flow ~'tometric technique. Further cell cycle analyses were performed by means ofpropidium iodide (PI) staining. Interestingly we found two separate populations of U266 based on the differences in their light scatter properties in the floxx~'tometer. One population had high scatter signals (HIGH SS) and the other low scatter signals (LOW SS). Most (75%) of the cells in the LOW SS cluster were positive for intracellular IgE while the expression of surface IgE was lower than that of the HIGH SS cluster. We further confirmed the immcellular location of IgE in the FOG treated U266 cells by means of immuuofluoresccnce microscopy analyses. In comparing the two clusters for expression of the plasma cell antigens CD38 and PCA-1 different patterus were observed. While the LOW SS cluster displayed a high (76%) expression of PCA-I and a low (5%) CD38 expression, the expression of the HIGH SS cluster was 21% and 42%, respectively. About 2/3 of the cells in the LOW SS cluster were in the G~ phase of the cell ~,cle while 1/3 were in the S/G2 phase. As to the HIGH SS cluster the opposite distribution (2/3 in S/G:) was observed. Taken together our results implicate an association between cell cycle stages, expression ofintraccllular IgE. and plasma cell markers. 463 rhGM-CSF promotes an antigen-presenting phenotype in human macrophages JJ Caulfield BSe. DM Kemenv PhD. 3"1-1 Lee MD UMDS, Guy's Hospital, London, UK Asthmatic airways are charaeterised by the presence of enhanced levels of GM-CSF and increased numbers of macrophages. Ja order to assess whether GM-CSF can modulate macrophage function, we have developed an in vitro assay in which monocytes isolated from human peripheral blood were cultured for seven days in RPMI + 50% AB serum in the presence or absence of rhGM-CSF (50 U/ml). The cultured monocyte-dedved macrophages (MDMs) increased in size and phagocytic ability and expressed antigens involved in co-stimulation and antigen presentation (CDIIa, CDIIb, CDIIc, CD54 and HLADR). After 7 days, monocytes and CD4s were isolated from the same donor. Proliferation assays were then performed in triplicate with lx104 freshly isolated monocytes or MDMs and lx105 CD4 cells/well with either HDM or PPD as antigen, for 6 days. CD4 proliferation was determined by [3H]-thymidine incorporation. In the absence of GM-CSF, CD4 proliferation was reduced when HDM or PPD was presented by MDMs, compared to the values obtained with freshly isolated monocytes. In the presence of GM-CSF, CD4 proliferation was comparable to that observed with monocytes when the respective antigen was presented by MDMs. This data suggests that GM-CSF may direct macrophage differentiation into an antigen-presenting, rather than a suppressive, phenotype in the asthmatic lung.