J.H. Gibbs

Lymphocyte subsets and Langerhans cells in allergic and irritant patch test reactions: histometric studies

This study has attempted to distinguish between allergic and irritant reactions to patch tests by semiquantitative histological methods. The extent of perivascular chronic inflammatory infiltrate at 72h in irritant patch test reactions to sodium lauryl sulphate was shown to be small and very consistent. whereas in allergic reactions in nickel sulphate it was generally larger and more variable in size (p < 0.02). The two major lymphocyte subsets (T4 and T8) were randomly intermixed in both types of reaction and formed the major component of both the perivascular and diffuse dermal infiltrate, without any evidence of selective migration The T4:T8 ratios were similar in focal and diffuse infiltrates. The number of T6 dendritic (putative Langerhans) cells in the epidermis (per mm inner epidermal length) was usually greatly reduced in irritant reactions (5–16 mm −1, mean 10 mm−1) but remained within normal limits in allergic reactions (6–33 mm−;1, mean 21 mm–1) (p <0.001). Comparable results were seen with other irritants (mercuric chloride and benzalkonium chloride) and older allergens (neomycin sulphate. ethylene diamine and potassium dichromate).

Histometric study of the localisation of lymphocyte subsets and accessory cells in human Mantoux reactions.

Intradermal injection of purified protein derivative produced typical delayed type hypersensitivity reactions in five healthy human subjects. The major subpopulations of lymphocytes and certain accessory cells were located in frozen sections of biopsies of the lesions with monoclonal antibodies and immunohistochemical staining. The densities (expressed as number/unit area for comparison) of the different types of cells were counted at various microanatomical locations in the tissue. The inflammatory cells were concentrated in narrow zones, initially (24 h) only surrounding small blood vessels but later (48-96 h) also around sweat ducts. Lymphocytes were the predominant cell type at these sites with T4 and T8 cells randomly intermixed at a ratio similar to that in the mononuclear cell fraction of the peripheral blood samples removed at the time of biopsy. There was also a scanty diffuse infiltrate in the intervening dermis, but here the T4:T8 ratio was significantly lower than that in the peripheral blood or perivascular cuffs. There was considerable intersubject variation in the relative preponderance of T8 cells in the diffuse infiltrate. The results suggest that there is no subset selection in the initial emigration of lymphocytes through vascular endothelium in the delayed hypersensitivity reaction, but that the subsets behave differently during the subsequent migration through the tissues. It remains to be determined whether the extent to which T8 cells migrate more rapidly than T4 cells through the tissues may influence the reaction at the site of entry of organisms or antigens into the body by altering the balance of the immunoregulatory lymphocyte subsets. This may underlie some of the differences in susceptibility to infection between subjects and determine the type of granuloma that develops in a particular patient.

A new method of testing for mitogen-induced lymphocyte stimulation: measurement of the percentage of growing cells and of some aspects of their cell kinetics with an electronic particle counter.

Human lymphocytes were cultured with or without added PHA for periods not exceeding one day so that none of the cells had entered the first mitotic division. The size distribution of the cells was measured with an electronic particle counter and the results were collected in a multichannel analyser. An iterative stochastic model was developed to estimate the proportion of responding cells and their growth characteristics. This mathematical model was based on a few simple assumptions about the pattern of cell growth and was considered to fulfill basic requirements of plausibility and parsimony. The technique described in this paper makes measurement of the absolute percentage of responding cells and their average growth rate in mitogen-induced lymphocyte stimulation tests possible in routine diagnostic laboratories for the first time.

Measurement of skin swelling in the tuberculin test by ultrasonography

A new method is described for non-invasive measurement of the thickness of the skin at the site of a skin test from the echo pattern of a pulsed ultrasonic (15 MHz) A-mode scanner. In tuberculin tests on normal volunteers, the skin thickness increased rapidly during days 1 and 2 and was usually greatest at day 4. The echogram measurements can be used to calculate the increase in skin volume and this is disproportionately greater than would be expected from measurements of the diameters of erythema and induration.

Dose-related depression of pha-induced stimulation of human lymphocytes by hydrocortisone

Abstract Hydrocortisone inhibits PHA-induced lymphocyte stimulation by reducing the number of cells entering G1-phase and by slowing the volume growth rate during the first 24 h of culture: the kinetics of cellular recruitment is unmodified. This inhibition has a linear log-dose response curve and there is marked intersubject variation in normal adults. Pre-incubation of the cells with hydrocortisone greatly potentiates its inhibitory effect: when added after the start of PHA stimulation, hydrocortisone becomes progressively less effective until it has lost most of its activity by 5 h, even though the cells do not show any microscopic evidence of having entered the G1-phase at that time. The experiments have elucidated the mode of action of hydrocortisone inhibition on the early stages of PHA-induced lymphocyte stimulation and shown an unexpectedly large intersubject variation in responsiveness that might have therapeutic significance. The methods that have been employed could be adapted for clinical monitoring of the immunosuppressive effect of glucocorticoids in individual patients and for screening synthetic steroids for glucocorticoid activity.

A simple method for determining the extent of cellular contamination in peripheral blood lymphocyte preparations

A method is described for calculating, from the size distribution of the cells, the extent of cellular contamination of lymphocyte concentrates prepared from venous blood. The precision of this method was checked by direct comparison with differential leucocyte counts obtained by direct microscopy on 29 samples chosen because they showed a wide range of intensity of contamination. By virtue of its technical simplicity, the method has proved useful in a diagnostic service laboratory for checking the purity of lymphocyte preparations either before performing lymphocyte function tests or after a period of 24 h in tissue culture.

Measurement by Quantimet 720 of the proportion of actively growing cells in tissue cultures of human lymphocytes

The size‐distribution of normal human lymphocytes growing in tissue culture was measured with a Quantimet 720 image‐analysing computer. The proportion of cells undergoing volumetric growth after phytohaemagglutinin (PHA) stimulation was derived from a simple mathematical model describing the growth patterns of the cells up to the first mitotic division. The approach may have general application in biology as a method for measuring the proportion of growing cells by microscopy.

Serum Inhibitory Factor in Lepromatous Leprosy: Its Effect on the Pre-S-Phase Cell-Cycle Kinetics of Mitogen-stimulated Normal Human Lymphocytes

The sera of ten Egyptian men with long‐standing lepromatous leprosy (LL) (mean duration 17.4 years) that had failed to respond in dapsone treatment were shown to inhibit mitogen stimulation responses of normal human lymphocytes. When first tested, the sera partly inhibited the response to phytohaemagglutinin (PHA) and pokeweed mitogen and virtually abolished that to concanavalin A (Con A): after repeated freezing and thawing, the Con A inhibition had disappeared, whereas the PHA response was still partly Inhibited. The inhibitory serum factor(s) had similar actions on lymphocytes from each of six normal donors. Although the sera varied in potency, they showed similar dose response curves when tested against lymphocytes from a single donor. The principal action of the sera was to reduce the number of cells responding to mitogen, without modifying the kinetics of recruitment or rate of volume growth during G1‐phase in those cells that were unaffected by the inhibitory substances(s). Study of PHA dose‐response curves and of the effect of delayed addition of LL serum suggested that the serum factor(s) act by diminishing the responsiveness of the cells, rather than by reducing the concentrations of free mitogen or by blocking cell membrane mitogen receptors The serum from one apparently healthy attendant, who had nursed leprosy patients for 30 years but who did not have leprosy or other chronic infective disease, inhibited completely stimulation by all three mitogens in a manner different from that of LL sera. Serum from the other 13 control patients did not modify the response of normal lymphocytes to stimulation by any of the three mitogens studied. It was concluded that the inhibitory factor(s) in the scrum of patients, with LL were a consequence of the disease and not of the environment in which the patients lived. Microscopy confirmed that the techniques used for recovery of the cultured cells did not introduce bias into the volume spectroscopy measurements.

Development of asynchrony in growth of normal human lymphocytes during first day of culture after PHA stimulation

Abstract The growth in volume of human peripheral blood lymphocytes after PHA stimulation was measured with an electronic particle counter. The percentage of growing cells and average values describing growth during the elapsed period of culture were estimated by fitting, to the observed data, volume distributions derived from a mathematical model based on simple, plausible and parsimonious assumptions. Most of the responding cells were recruited between 9 and 18 h after the start of culture. The averaged growth rate of the stimulated cells did not change materially between 18 h and the end of the experiment (36 h). Progressive recruitment over the first 18 h of culture is probably the main cause of the asynchronous growth encountered when such cultures are sampled during continuous replication with T c previously estimated at 18–30 h. This conclusion was supported by computer simulation experiments.

Mechanisms of phytohaemagglutinin (PHA) stimulation of normal human lymphocytes: ‘trigger’, ‘push’ or both?

Abstract. The growth in volume of human peripheral blood lymphocytes after stimulation with various concentrations of PHA was measured with an electronic particle counter. The percentage of growing cells and averaged values describing their growth rates during the elapsed period of culture were estimated by fitting to the observed data the volume distributions derived from a mathematical model. With sub‐optimal doses, the percentage of cells stimulated, and their incremental growth rate, increased with increasing dose of PHA, but the time‐course of recruitment into the G1‐phase was similar with all PHA concentrations studied. The results provide strong support for the ‘trigger’ hypothesis that there is a distribution of stimulation thresholds within the lymphocyte population: consequently, increasing mitogen concentration will be expected to result in increased numbers of responding cells within the suboptimal concentration range.