Cell-mediated immunity can be detected in vitro by various reactions, many of which are mediated by soluble factors. The leukocyte adherence inhibition (LAI) test has been recently developed as a rapid in vitro test which may be a correlate of delayed-type hypersensitivity. LAI is a reaction in which leukocytes from sensitized donors, stimulated with the corresponding antigen in vitro, exhibit a decreased adherence to solid surfaces. The following studies were aimed at elucidating the mechanism of LAI.
Contact hypersensitivity in mice to picryl chloride (PCl) was chosen as a suitable immunological system for investigating the cell types reacting with the specific antigen in vitro. After direct (1-stage) LAI tests were used initially to determine the optimal antigen concentration, the LAI effect was subsequently demonstrated in indirect (2-stage) assays. Supernatants were obtained from sensitized spleen cells (taken 7 days after sensitization) after in vitro incubation with antigen for 60 min, and tested on a population of normal indicator cells. The supernatants contained a soluble lymphokine-like factor (leukocyte adherence inhibition factor—LAIF) responsible for the final reduction in cellular adherence. This 2-stage activity (as opposed to the 1-stage activity) was poorly reproducible. It also appeared that "late" spleen cells (taken from mice at times greater than 7 days after sensitization) were almost never active in LAI assays, whereas cells taken before this time showed variable LAIF production.
Sensitized spleen cells were fractionated on glass-bead or nylon-wool columns to determine the role of adherent cells (macrophages) in LAI phenomena. Glass-bead columns depleted macrophages (and B lymphocytes also to a slight extent), but under the particular conditions used, the nylon wool did not appear to deplete B lymphocytes. It was found that LAI activity of cells in 2-stage assays was abrogated by glass-bead columns, and that addition of a macrophage source (normal peritoneal cells or corresponding macrophage-enriched cells) to the resulting non-adherent cells allowed LAIF to be produced. This implied that macrophages were required for LAIF production in this system. Either syngeneic or allogeneic macrophages were effective in restoring the LAI response of glass-bead non-adherent cells. The reconstitution of LAI activity was observed regularly even with cells showing no initial activity, and with cells taken at times greater than 7 days after sensitization. Such effects were interpreted as being due to the concomitant removal, by the glass-bead columns, of cells suppressive for LAIF production along with the macrophage population. This was confirmed by demonstrating that glass-bead adherent cells could suppress LAIF production by unfractionated cells, in an antigen-specific manner. Passage of sensitized spleen cells through nylon-wool columns did not remove this suppressor cell fraction.
Affinity columns were used to separate T and B lymphocyte classes from spleen cell populations, after initial depletion of macrophages on nylon wool. It was found that both of these major lymphocyte classes could produce LAIF, but only in the presence of normal peritoneal cells (macrophages). Moreover, LAIF activity was not seen with mixtures of T and B cells, indicating a mutual suppressive effect. The B cell LAI activity was resistant to anti-Thy-1.2 serum treatment, and treatment of unfractionated spleen cells with the same antiserum allowed LAI effects to be seen with initially unreactive cells. This confirmed the B cell LAIF production, and indicated that T cells were suppressing this B cell activity in the unfractionated populations. The B suppressor cells, the T suppressor cells, and the B cell subpopulation active in LAIF production, were glass-bead adherent. The LAIF-producing T cells were non-adherent. Suppressive phenomena did not extend to non-specific suppression of lymphocyte blastogenesis, as stimulation of sensitized spleen cells by concanavalin A (Con A) proceeded just as effectively as with normal spleen cells.
LAI activity in vitro was compared with in vivo adoptive transfer of delayed-type hypersensitivity. The in vitro data were found to correlate with the in vivo test, since "late" sensitized spleen cells could not effect adoptive transfer until they had been passaged through glass-bead columns. The glass-bead adherent fraction suppressed adoptive transfer by "early" cells (taken 4 days after sensitization) in an antigen-specific manner. This strengthened the viewpoint that LAI tests are correlates of delayed-type hypersensitivity.
The second major approach toward the study of LAI mechanisms was to attempt to characterize the soluble factors involved. Con A was initially used to stimulate normal spleen cells in the hope of obtaining large amounts of LAIF. It was found that Con A elicited both LAIF and a presumptive adherence stimulation factor. Both of these factors could also be demonstrated from antigen-stimulated cells under the correct conditions. The concentration of serum in the initial cell-antigen mixture appeared to be the critical factor in determining whether LAI or adherence stimulation effects were seen, and a high concentration of serum in the indicator cell^ supernatant mixtures was also necessary to demonstrate both antigen-stimulated and mitogen-stimulated LAIF. Supernatants obtained after stimulation of spleen cells by both Con A and antigen were fractionated on Sephacryl S-200. The mitogen-induced LAIF and adherence stimulation factor had molecular weights of 26-52,000 and 51-96,000 daltons respectively, whereas the specific antigen-induced LAIF and adherence stimulation factor had molecular weights of 44-≥96,000 and >113,000 daltons. This indicated that adherence-modulating factors, especially those from different sources, were heterogeneous. Con A-stimulated LAIF appeared to be sensitive to high pH and neuraminidase, and the corresponding adherence stimulation factor was also pH sensitive. Antigen-stimulated LAIF activity was abrogated by the presence of N-acetylglucosamine in the indicator cell-supernatant mixtures, indicating a role for this monosaccharide as a component of the LAIF receptor. Supernatants from PCl-sensitized T and B cells were passaged through anti-mouse γ-globulin affinity columns, which appeared to remove B cell LAIF, but not T cell LAIF. This demonstrated that these T and B cell factors were dissimilar.
The general conclusions were that, especially with spleen cells from contact sensitized mice, both the cellular generation and the detection of LAIF were complex, with suppressive cellular interactions and adherence stimulation modulating the phenomenon of LAI. The physical nature of the soluble factors involved with LAI effects also appeared to be complex. Results obtained from LAI assays were correlated with observations on in vivo cell-mediated immunity.