Subject(s)
Ampicillin/pharmacology , Lymphocytes/drug effects , Receptors, Fc/drug effects , Animals , Humans , Sheep/immunologyABSTRACT
A flow-cytofluorometric method, based on the differential stability of deoxyribonucleic acid versus ribonucleic acid with the metachromatic dye, acridine orange, simultaneously measures the following parameters of stimulation in mixed lymphocyte cultures: (a) number of nonstimulated cells; (b) total number of stimulated lymphocytes; (c) number of stimulated lymphocytes in G1, S and G2 + M phases of the cell cycle; (d) number of macrophages; (e) number of dead cells. The progress of lymphocyte stimulation may also be measured by a parameter representing ribonucleic acid accumulation per cell. The method is rapid, avoids cell rinsing, fixation and centrifugation and is applicable to microcultures. Multiparameter analysis of cell stimulation which provides simultaneous measurements of lymphocyte proliferation and accumulation of ribonucleic acid per cell may prove to be a more sensitive assay of histocompatibility than tests based only on cell proliferation (tritiated thymidine incorporation).
Subject(s)
DNA/analysis , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Lymphocytes/analysis , RNA/analysis , Acridines , Cell Division , Macrophages/analysis , Methods , Spectrometry, FluorescenceABSTRACT
Leukocyte migration inhibitory factor (LMIF) production in mixed lymphocyte culture (MLC) reactions is the result of cellular interactions based on two separate phenomena: the capacity of lymphocytes to stimulate in MLC, and the capacity of lymphocytes to respond in MLC. Puromycin-treated lymphocytes are capable of stimulating allogeneic cells for LMIF production, but are unable to respond with synthesis of LMIF (one-way MLC-LMIF test). We have studied the stimulating and responding capacity of lymphocytes from patients with different immunodeficiency syndromes in a one-way MLC-LMIF assay. Lymphocytes from patients known to have qualitative and quantitative defects of T cell or B cell functions (Hodgkin's disease, mycosis fungoides, thymoma, chronic lymphatic leukemia) were found to respond poorly as measured by mediator production although their stimulating fuction was frequently retained. Patients with advanced solid tumors often had both MLC-stimulating and responding functions depressed. There was no apparent correlation between mitogen response and MLC-induced LMIF responses or between MLC proliferative response (as measured by thymidine incorporation) and mediator production. Studying of stimulatory and responding capacity of lymphocytes in the MLC-LMIF assay provides a new tool for assessing immunocompetence and allows for in vitro evaluation of cellular interactions that may play an important role in vivo.
Subject(s)
Immunologic Deficiency Syndromes/immunology , Lymphocyte Culture Test, Mixed , Lymphocytes/metabolism , Lymphokines/metabolism , Candidiasis, Cutaneous/immunology , Female , Hodgkin Disease/immunology , Humans , Leukemia, Lymphoid/immunology , Male , Mycosis Fungoides/immunology , Neoplasms/immunology , Thymoma/immunology , Thymus Neoplasms/immunologyABSTRACT
Lymphocytes from patients with primary and secondary immunodeficiency disease were tested for capacity to produce LMIF after mitogen and antigen stimulation as well as for ability to stimulate and respond in unidirectional MLC-LMIF assay. Different patterns of immune abnormality in vitro were detectable when Con A and Candida albicans antigen were used. In addition, significant abnormalities in LMIF responding and stimulatory capacity were demonstrated in patients with Hodgkin's disease. LMIF production after stimulation with different agents allows for a better characterization of cellular defects in immunodeficiency disease.
Subject(s)
Immunologic Deficiency Syndromes/immunology , Macrophage Migration-Inhibitory Factors/analysis , Antigens, Fungal , Humans , Lymphocyte Activation , Lymphocytes/immunology , MitogensABSTRACT
Puromycin treatment of lymphocytes was used to develop a one-way test for leukocyte migration inhibitory factor (LMIF) production in the mixed lymphocyte culture (MLC) reaction. Lymphocytes incubated with this protein synthesis inhibitor induced a vigorous mediator production by nontreated allogeneic cells, being themselves unable to respond to stimulator cells. When puromycin-treated cells were stimulated with the mitogens PHA, ConA, or PWM, overall protein and DNA synthesis were significantly decreased with concomitant abolishment of LMIF production. Viability of stimulator lymphocytes was found to be essential for generation of the mediator in MLC reaction.
Subject(s)
Lymphocytes/immunology , Macrophage Migration-Inhibitory Factors/biosynthesis , Cell Survival/drug effects , DNA/biosynthesis , Hot Temperature , Humans , Lymphocyte Culture Test, Mixed , Lymphocytes/drug effects , Lymphocytes/metabolism , Protein Biosynthesis , Puromycin/pharmacologyABSTRACT
A standardized microculture system has been developed to assess the ability of lymphocytes to secrete leukocyte migration inhibitory factor (LMIF) in response to the nonspecific mitogen concanavalin(Con A). LMIF-rich supernates collected from stimulated lymphocytes cultured in plastic microtiter plates are assayed by pulse exposure of purified human granulocytes and inhibition of their migration in agarose medium. LMIF activity in this system is suppressed by the protein synthesis inhibitor puromycin, but not by inhibition of lymphocyte proliferation by irradiation. It is demonstrated that normal lymphocytes stimulated with mitogen elaborate LMIF activity, while lymphocytes from malignant lymphoma patients are frequently unable to produce it. Thus, mitogen-induced mediator production may be a useful parameter in further characterization of primary and secondary immunodeficiencies.
