Adi A. Gerblich

Changes in T-Lymphocyte Subpopulations after Antigenic Bronchial Provocation in Asthmatics

To determine whether inhaled agents can alter T-cell subsets in the peripheral blood of patients with bronchial asthma, we tested six asymptomatic asthmatics who were sensitive to mixed grass (positive skin test) with mixed grass extract, methacholine, and an antigen to which they were not sensitized (negative skin test). Levels of OKT4 cells (helper T lymphocytes) were reduced in the peripheral blood immediately after the challenge with mixed grass extract, and remained low for at least 72 hours. Levels of Ia-positive (activated) T cells were increased 48 hours after the challenge. No changes were observed in any of these T-cell subpopulations after challenge with methacholine or after the inhalation of an equal amount of an antigen to which the subjects were not sensitized. These results suggest that the selective loss of circulating helper T cells and an increase in activated T cells after an asthmatic attack induced by antigenic inhalation may serve as an indicator of immune-mediated bronchoconstriction.

Atopic asthma: T lymphocyte subpopulations

Atopy is associated with diminished cell‐mediated immunity and increased amounts of IgE, both of which may be caused by imbalances of T lymphocyte subsets.

A proposed standardized method for bronchoprovocation tests in toluene diisocyanate-induced asthma

Exposure to vapors of toluene diisocyanate (TDI), a chemical widely used in the plastics and rubber industry, can cause respiratory disease at low concentrations and can be a nonspecific respiratory irritant at high concentrations.‘. 2 While these clinical patterns have been known for some time, laboratory diagnosis has been a challenge because of several problems, including lack of methods for performing quantitative studies of the effect of inhaling precise TDI concentrations. For this to be possible, a delivery system must produce adjustable, stable concentrations of TDI that can be monitored accurately throughout the time of exposure to TDI fumes. Three major problems are encountered in the implementation of a suitable delivery system. First, TDI is an extremely volatile liquid at room temperature which readily produces a dense vapor (vapor density = 6.0; air = 1). Thus, exposed TDI liquid can create vapors of high concentration which vary with location, room size, and room ventilation characteristics. Second, TDI vapor readily adsorbs onto the surface of most materials, except glass and Teflon,” so that efficient transport of TDI test gases is difficult. Third, the present commercially available instruments for the continuous measurement of TDI concentration have an overall response time of 18 min. Therefore, unless the delivery system is stable throughout test procedures, it is impossible to determine the actual concentration of TDI the subject is inhaling because one is always measuring 18 min behind the event. Previous methods of controlling inhaled dosages of TDI for challenge-response studies have been based on reproducing a given environmental situation. Pepys et al.” had their subjects tested in a closed room, in which a polyurethane varnish containing

Regulation of the in vitro anamnestic antibody response by cyclic AMP: IV. Evidence for participation of prostaglandins of the E series in the early events

Abstract The addition of KLH to KLH-primed rabbit lymph node cell cultures induced an anamnestic antibody response. The further addition of prostaglandins of the E series, but not PGF1 α , enhanced this antibody response manifold. The addition to these cultures of prostaglandin synthetase inhibitors together with KLH inhibited antibody production. At the concentration (10 −4 ) required to inhibit antibody synthesis, by a variety of criteria one of these inhibitors, indomethacin, was shown not to exert its effects through cytotoxicity. By contrast, two other inhibitors of prostaglandin synthesis, Ro-20-5720 and Ro-3-1314, inhibited antibody synthesis because of their cytotoxicity. The inhibition of the antibody response by indomethacin did not occur when PGE1 or PGE2 was added concurrently to these cultures, clearly showing that inhibition was due to a deficiency of prostaglandins. These findings strongly suggest that induction and/or regulation of the in vitro anamnestic antibody response of KLH-primed lymph node cells to 1 and 100 μg KLH requires continued prostaglandin synthesis. Potential mechanisms for the regulation of the antibody response by prostaglandins are discussed.

Antigen-induced T-cell changes: modulation by pharmacologic agents.

To determine the effect of pharmacologic modulation of alterations of peripheral blood T-cell subsets caused by antigen-induced bronchoconstriction, we administered albuterol immediately after antigen-induced bronchoconstriction in a double-blind to protocol to 12 atopic asthmatic subjects. We also administered cromolyn sodium before antigen to 7 of the same subjects. Peripheral blood T-cell subset composition (CD4, CD8, la) of a highly purified T-cell preparation was determined before, 24, 48, 72, and 168 h after bronchoconstriction.We found that placebo inhalation immediately after antigen-induced bronchoconstriction did not affect subsequent peripheral blood T-cell subset changes (decrease in CD4+ and increase in Ia+ T lymphocytes). In contrast, inhaled albuterol abolished these T-cell subset changes. Although cromolyn sodium significantly decreased the severity of antigen-induced bronchoconstriction, it did not affect T-cell subset composition changes at the dosage used.We conclude that albuterol can ablate T-cell subset changes associated with antigen-induced bronchoconstriction. Cromolyn sodium ameliorates bronchoconstriction, but has no affect on T-cell subset composition changes. This implies that T-cell changes and bronchoconstriction caused by antigen inhalation are mediated through different pathways.

Elastolysis of Insoluble Elastin

We developed an assay for measurement of elastolytic activity using insoluble 3H-labelled particulate elastin adherent to plastic that is capable of detecting 150 picograms of pancreatic elastase. This equals or exceeds the sensitivity of the most sensitive previously reported systems, without requiring sodium dodecyl sulfate treatment of the elastin. Elastin digestion is dependent upon substrate and elastase concentration, but is not linearly related to time. This is partially attributable to elastase denaturation or autolysis under the assay conditions. The assay could easily detect elastase secreted by either peritoneal or alveolar macrophages. Compared to previously described assays using substrates that closely resemble the physiologic substrate, this represents a considerable increase of sensitivity of detection of elastolytic activity of enzymes.

In vitro anamnestic immune responses and modulating factors

SummaryThe term “anamnestic” refers to the specific and enhanced immune responses of antigen-immunized (primed) lymphoid memory cells to secondary challenge with a foreign substance (antigen). These responses include the accelerated and quantitatively greater syntheses of antibody and other macromolecules than upon primary challenge of such cells. Rabbits were primarily immunized with keyhole limpet hemocyanin (KLH). Six days later their memory lymph node cells (LNC) were removed, and upon culture with KLH, responded with the synthesis of antibody, immunoglobulin (Ig), protein, DNA and RNA, as well as with active transport of dibutryl cyclic AMP (DbcAMP). Purified thymus-derived (T) LNC were prepared on anti-rabbit Ig affinity columns. Bursal-equivalent (B) cells were prepared by binding to a complex of sheep erythrocytes (SRBC)-antibody to SRBC-complement and centrifugation of these complexes on suitable gradients. When these T and B KLH-primed LNC were mixed and challenged with KLH the aforementioned macromolecular syntheses and active transport occurred. Indeed, by a variety of criteria, the reconstituted anamnestic immune responses were indistinguishable from these responses of unfractionated LNC. Antigenic stimulation of KLH-primed T cells induced the synthesis of proteins and DNA, but not antibody, but antigenic challenge of KLH-primed B cells did not evoke these syntheses. However, added KLH induced a mixture of T and B antigen-primed LNC to synthesize more protein, Ig, DNA than either population alone and more antibody than T cells per se; B cells required help for all of these responses. The thymus (T) cell-dependent phase of in vitro anamnestic antibody response lasted the first 24–36 hr.The antibody response was regulated by antigen-concentration. One μg KLH evoked maximal antibody synthesis, 10 and 100 μg KLH much less. Challenge of the separated T and B cell populations with different KLH concentrations, followed by recombination and eventual assay of antibody synthesis revealed different optima. The optimal concentration for T cell help was 0.01–0.1 μg KLH; higher amounts induced much less antibody production. The optimum for B cells was 1–10 μg KLH; 100 μg inhibited antibody formation.The antibody response to KLH and human serum albumin (HSA) was regulated nonspecifically utilizing LNC from rabbits immunized simultaneously with these two antigens. Thus stimulation of LNC from these rabbits with either antigen induced the synthesis of antibodies to both antigens. HSA and KLH did not cross-react either serologically or cellularly. Cross-stimulation of antibody synthesis also was observed when rabbit LNC were primed with KLH and Mb. However, in this instance, cross-reaction between KLH and sperm-whale myoglobulin (Mb) was observed at the cellular, presumably the T cell, level, although not at the antibody (B cell) level. The antibody response could also be modulated by exogenous cholera enterotoxin (CT), dibutyryl cyclic AMP (DbcAMP) and prostaglandins of the E series. The addition of each substance together with 1–100 μg KLH to KLH-primed LNC enhanced the antibody response many-fold. CT-induced non-immunized LNC to produce soluble factor(s) (SF) which, when added to KLH-primed LNC together with KLH, enhanced antibody synthesis significantly. The addition of Indomethacin, an inhibitor of PGE synthesis to KLH-immunized cells together with KLH inhibited antibody production, suggesting that PGE was involved in this response. Evidence was adduced that neither cyclic AMP nor PGE was required for the antibody response: Ca2+ was not required for induction of this response by KLH, but only its regulation by cAMP.Moreover, when KLH-primed LNC were fractionated on Nylon columns, the effluent cells were induced by KLH to synthesize antibody, but this synthesis was not enhanced by added DbcAMP or PGE; presumably, regulatory cells were removed on the column. Added KLH induced PGE synthsis in these cultures; this synthesis required macrophages. In all of the LNC cultures — including cultures from rabbits immunized with KLH, HSA, and MB months or a year earlier — much antibody synthesis occurred even when antigen was not added to the cultures. This “spontaneous” antibody was anamnestic, thymus (T cell)-dependent and involved the interaction of residual immunogen on dendritic cells with T and B memory cells. This spontaneous antibody response provides a model for the study of the factors involved in the longterm maintenance of humoral immunity.Mb was employed as a source of more refined antigenic determinants. Rabbits were immunized with Mb in complete Freunds adjuvant. The addition of small synthetic peptides corresponding to the five antigenic sites of Mb to the Mb-primed LNC induced the synthesis of antibody, Ig, protein, DNA, RNA, and macrophage migration inhibitory factor (MIF). The N terminal 1–6 peptide, which is not antigenic, i.e. does not combine with antibody to Mb, also induced all of these syntheses, except MIF. These peptide-induced responses appeared to be thymus-dependent.