Christian H. L. Rieger

Detection of intracellular cytokines by flow cytometry

During the last years it has become increasingly clear that production of most cytokines is not confined to one cell type. Thus, a method to detect cytokines at the single cell level would be a helpful tool to study the contribution of different cells to cytokine production in heterogeneous cell populations. Recently, Sander et al. (1991) demonstrated that it is possible to detect intracellular cytokines by fixation with paraformaldehyde, permeabilization with saponin and subsequent indirect immunofluorescent staining using fluorescence microscopy. Here, we describe a modified method to increase the specific intracellular staining which enables us to detect IFN-gamma, IL-2 and IL-4 producing cells by single laser flow cytometry. The carboxylic ionophore monensin was used to interrupt intracellular transport processes leading to an accumulation of the cytokine in the Golgi complex. This resulting increase of the signal/noise ratio permitted us to detect weakly fluorescent cells such as IL-4 producing cells. While IL-4 was detected in approximately 1-3% of peripheral mononuclear cells from healthy donors, up to 30% of the cells produced IFN-gamma and nearly 50% IL-2 after phorbol ester and ionomycin stimulation. Microscopic and flow cytometric analysis showed a highly significant correlation. Using three-color flow cytometry it was possible to measure intracellular cytokines and cell surface markers simultaneously. Subpopulations of human T cells (e.g., CD4+ CD45R0-) producing a restricted cytokine pattern could be identified by cell surface staining and were characterized by their cytokine production. Consequently, there was no further need for cell sorting to determine cytokine producing subsets in heterogeneous cell populations. We have tested human T cell clones for intracellular cytokine production and found a high concordance to ELISA analysis of the supernatants. We conclude that detection of intracellular cytokines by flow cytometry is a rapid, easy and semiquantitative assay which may be used to study individual cells in heterogeneous populations as well as to screen homogeneous cells for their cytokine pattern. This method is particularly relevant in view of the accumulating evidence of the functional role that subsets of (T) cells may play in various diseases depending on the pattern of cytokines they produce.

Decreased frequency of interferon-γ– and interleukin-2–producing cells in patients with atopic diseases measured at the single cell level

BACKGROUNDnRecently, diminished interferon-gamma (IFN-gamma) and increased interleukin (IL-4 production in peripheral blood mononuclear cells (PBMCs) from atopic patients have been described by several groups, measured as total cytokine content in culture supernatants. These studies suggested a predominance of TH2-like cells producing large amounts of IL-4 in atopic patients. It is not clear whether the reported cytokine imbalances are the result of an alteration in the distribution of specific T-cell subsets or whether intrinsic dysregulation in cytokine production is a characteristic of atopic individuals.nnnOBJECTIVEnThis study examined the production of IFN-gamma, and IL-2 in PBMCs from atopic patients at the single cell level with the use of freshly isolated lymphocytes.nnnMETHODSnWe recently described a flow cytometric assay in which three-color analysis was used to study the production of a cytokine of interest in a T-cell subpopulation defined by two cell surface markers. PBMCs from 23 atopic patients and 14 control subjects were stimulated with phorbol ester and ionomycin for 5 hours. PBMCs from seven patients and seven control subjects were also cultured with immobilized anti-CD3 antibodies for 24 hours. Cells were fixed, made permeable, and stained for intracellular cytokines in combination with cell surface markers CD3, CD8, and CD45RO. Cytokine-producing cells were analyzed by gating on T-cell subsets.nnnRESULTSnIFN-gamma-producing cells were significantly decreased (p < 0.05) in CD4+ T cells but not in CD8+ T cells of atopic patients. CD45R0+ and CD45R0-T cells showed a decreased proportion of IFN-gamma-producing cells (p < 0.05 and p < 0.01, respectively). IL-2 production was diminished in all T-cell subsets (p < 0.01). The number of IL-4-producing cells was not elevated, and such cells were exclusively found in the CD45RO+ T cells. Analysis of culture supernatants of sorted CD45RO+ T cells for IL-4 and IFN-gamma production confirmed these results.nnnCONCLUSIONnOur findings provide evidence that a reduced IFN-gamma production in atopic patients is due to an intrinsic defect selectively found in the CD4+ T cells. Because IL-2 production was markedly decreased but IL-4 production was unchanged, our data demonstrate a deficiency in the ability of atopic T cells to produce TH1-like cytokines on stimulation with phorbol ester, ionomycin, or anti-CD3 monoclonal antibodies.

Effects of SO2 exposure on allergic sensitization in the guinea pig

The effect of sulfur dioxide (SO2) exposure on local bronchial sensitization to inhaled antigen was studied in the guinea pig. Exposure to SO2 (0.1 to 16.6 ppm) was performed in a 20 L exposure chamber for 8 hours on 5 consecutive days, while temperature, moisture, and concentration of SO2 were monitored and kept constant. SO2 concentrations were measured hourly by Schiffs reaction. On the last 3 days, SO2 exposure was followed by inhalation of nebulized ovalbumin (OA) for 45 minutes. One week later, specific bronchial provocation with inhaled OA (0.1%) followed by plethysmographic measurements of airway obstruction were performed every 2 days during a 2-week period. Specific antibodies against OA were measured in serum and bronchoalveolar fluid by a direct enzyme immunoassay. The SO2-exposed group (N = 17) demonstrated 67% to 100% positive bronchial reactions to inhaled OA, depending on the concentration of SO2, whereas the control group without previous SO2 exposure (N = 14) demonstrated bronchial reactions in only one animal (7%: p less than 0.05). The degree of bronchial obstruction was significantly higher in the exposed group, compared to the control group, for all SO2 concentrations (p less than 0.05). OA-specific antibodies in serum and bronchoalveolar fluid increased in SO2-exposed groups significantly, compared to the control group (p less than 0.05). It is concluded from these results that exposure to SO2 in low and medium concentrations can facilitate local allergic sensitization in the guinea pig.

Levels of Antibodies Specific to Tetanus Toxoid, Haemophilus influenzae Type b, and Pneumococcal Capsular Polysaccharide in Healthy Children and Adults

ABSTRACT Antibody levels specific for capsular polysaccharides of Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) and for tetanus toxoid were measured in serum samples of 386 age-stratified subjects. The study group consists of healthy adult blood donors and hospitalized children undergoing elective surgery, excluding individuals with a history of infection. In children, anti-tetanus toxoid antibody levels displayed two peaks of 1.20 IU/ml (20.4 mg/liter) and 1.65 IU/ml (28.1 mg/liter) related to the schedule of routine childhood immunization in the first year and at 8 years of age. Eighty percent of the antibodies are of the immunoglobulin G1 (IgG1) isotype. For pneumococcal capsular polysaccharide (PCP), the specific antibody levels represent the acquisition of natural immunity. The initial concentration of 9.2 mg/liter was low in infancy (0.5 to 1 years of age) and remained low until 3 to 4 years of age (14.6 mg/liter). During this period PCP antibodies were almost 100% of the IgG2 subclass. Thereafter, IgG anti-PCP antibody titers increased steadily to adult levels (59.5 mg/liter). The data are intended to provide reference ranges to aid in the interpretation of specific antibody determinations in the clinical setting.

Development of the capacity to produce specific antibody to an ingested food antigen in the premature infant

Thirteen premature infants were given bovine serum albumin, a cow milk protein, by addition to their formula. Serum antibodies to BSA developed in three infants 36-38 weeks gestation, confirming that exposure to the antigen in the gastrointestinal tract will immunize infants born after 36 or more weeks gestation. Serum antibodies to BSA, however, were detected in only one of two infants of 35 weeks and in none of eight infants of 30-34 weeks gestation. The results show that the capacity to make specific antibodies to BSA develops around 35-36 weeks gestation, despite the prior appearance of organized lymphoid tissue and independent of antigen exposure.

Rotavirus infection and bradycardia-apnoea-episodes in the neonate

Rotavirus (RV), a common cause of infectious enteritis in young children including neonates, has not been associated with central nervous symptoms in standard textbooks. However, involvement of the CNS has been reported recently in case reports and small series. From 786 neonatal admissions in 1991 we retrospectively analysed the records of 215 inpatient neonates (68 preterm and 147 term infants) who developed diarrhoea during their stay on the neonatal ward and in whom stools were investigated for RV antigen by ELISA. All 215 neonates were continuously monitored for bradycardia-apnoea-episodes (BAE) at least 2 days before and during the entire diarrhoeal period. In neonates with RV antigen in stools (n=114) we found a higher incidence of BAE compared to neonates with RV negative stools (33% vs 8%,P<0.001 for bradycardia; 7% vs 0%,P<0.05 for apnoea). Furthermore, bradycardia episodes of RV positive neonates were more often followed by cyanosis (11 vs 0%,P<0.05) and intervention was more often necessary (31 vs 14%,P<0.05) than in the RV negative neonates.

Reconstitution of T-cell function in severe combined immunodeficiency disease following transplantation of early embryonic liver cells

In a 51/2-month-old male infant with adenosine deaminase-positive severe combined immunodeficiency disease, who had no suitable bone marrow donor, immunologic reconstitution was attempted with lymphoid cells obtained from the liver of a 4- to 5-week-old-male human embryo. A mild graft-versus-host reaction began three weeks later. T-cells, which were absent prior to infusion of hepatic lymphoid cells, rose to a maximum of 554/mm3 at 16 weeks post transplantation. A normal lymphocyte response to pokeweek mitogen was not present until 25 to 30 weeks and to allogeneic cells until 39 weeks. Postive in vitro lymphocyte responses to Candida albicans were found repeatedly after 52 weeks. Twenty months following transplantation the patient is free of clinical infection, although he requires regular injections of gamma globulin.

Oral Immunization to Milk Protein in Human Infants in the Presence of Passive Antibody

Summary: The possible influence of maternal antibody on the immune response to bovine serum albumin (BSA), a normal cows milk protein, was investigated in fullterm human neonates. Antibody production to BSA of 12 infants with passively acquired anti-BSA (Group I) was compared to the immune response of nine infants without passive anti-BSA at birth (Group II) during the first 6 months of life. All infants were raised on commercial cows milk formulas containing BSA in concentrations from 0.4–4.0 mg/dl. From 4 wk of age concentrations of circulating anti-BSA as measured by radioimmunoassay and enzyme-linked-immunosorbent-assay (ELISA) were higher in Group II, but differences were not statistically significant. There was no difference in the immune response between infants ingesting formulas with high BSA content compared to infants ingesting low concentrations of antigenic BSA. The main isotype associated with anti-BSA formation was IgG. IgA in measurable amounts appeared later and accounted for approximately 10% of circulating antibody in both groups at 6 months of age. Only small amounts of IgM- and IgE-anti-BSA were detected.

Alteration of T cell function in healthy persons with a history of thymic x-irradiation.

The possible late effects of x-irradiation to the infantile thymus were investigated by studying immune functions in 12 healthy persons with a history of thymic x-irradiation and healthy control subjects. No differences were found in serum immunoglobulin values, humoral antibody levels, lymphocyte counts, and lymphocyte reactivity to phytochemagglutinin, vaccinia virus, purified protein derivative (PPD), and allogeneic cells. The irradiation group exhibited cellular hyperresponsiveness to streptoskinase-streptodornase (SK-SD). In contrast, mean skin and in vitro lymphocyte responses to Candida albicans were depressed in the patients with thymic irradiation. A dissociation of these two Candida responses was found in only 1 of 14 healthy control subjects but in 7 of 12 irradiated individuals. While thymic irradiation did not result in impaired immunologic defenses leading to clinical disease, it caused alterations in T cell responses similar to those reported in patients with chronic mucocutaneous candidiasis.

Local antibodies to alpha-casein and beta-lactoglobulin in the saliva of infants.

ABSTRACT. Salivary antibodies were studied in 112 infants between 1 day and 8 yr of life. SIgA anticasein was present from birth in breast-fed and bottle-fed infants. Bottle-feeding resulted in significantly higher concentrations of SIgA anticasein at 3 wk to 3 months of life as compared to breast-feeding. Salivary anticasein declined toward the end of the 1st yr and was present in less than half of the children older than 1 yr. Salivary anti-lactoglobulin was also present at birth in some infants. Levels increased slightly over the following 3 months but remained low. Only a minority of older children had this antibody in their saliva.