Influence of fibronectin-containing blood products on lymphocyte reactivity

Two fibronectin (FN)-containing blood products, human peripheral blood plasma and cryoprecipitate, were examined for their effect on mitogen-induced lymphocyte transformation in vitro. Responses of human peripheral blood lymphocytes to phytohemagglutinin (PHA) were depressed in the presence of a plasma concentration above that required for maximum DNA synthesis, and this concentration must be present in cultures prior to lymphocyte activation. The removal from the plasma of heparin-induced cryoprecipitate, a complex consisting of FN, heparin, and fibrinogen, resulted in a significant reduction in the inhibitory effect of the plasma on the PHA response. Plasma specifically depleted of FN by affinity chromatography on gelatin-agarose beads was 32 percent less inhibitory to the PHA-induced stimulation of cells than untreated plasma; the remaining inhibitory activity in the FN-depleted plasma samples was attributed to the presence of other normal immunosuppressive factors. The inhibitory capacity of FN in plasma was similar to that obtained with purified FN alone, which indicates that, unlike that of other known plasma inhibitors, the immunosuppressive activity of FN was not altered by the presence of other components of plasma. Cryoprecipitate used in the treatment of hemophilia contains high levels of FN, and, as anticipated, PHA-induced lymphocyte transformation was markedly depressed in the presence of solubilized cryoprecipitate. The contribution of FN to the T-cell abnormalities in patients chronically receiving cryoprecipitate and/or factor VIII concentrates derived from cryoprecipitate warrants further investigation.     

Impaired Lymphocyte Transformation in Hodgkin''s Disease. EVIDENCE FOR DEPLETION OF CIRCULATING T-LYMPHOCYTES

The kinetics of lymphocyte transformation induced by phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were studied daily, with blood lymphocytes from normal individuals and from untreated patients in all stages of Hodgkin's disease (HD). In addition, spleen lymphocytes and lymph node lymphocytes were studied with similar techniques.Peripheral blood lymphocyte transformation stimulated by PHA was found to be depressed in all patients with HD (including those with localized disease and no symptoms) when small numbers of lymphocytes were cultured and studied during a 7-day period. Most patients with HD had an increased number of cells circulating in their blood which were actively synthesizing DNA. HD lymphocytes which demonstrated the highest initial rate of spontaneous DNA synthesis usually did not respond to PHA stimulation.Blood lymphocytes from normal individuals responded equally well to PHA and PWM in our system. HD blood lymphocytes consistently responded better to PWM than to PHA, with the response to PWM frequently within the normal range. Unless the spleen was extensively infiltrated with HD, spleen lymphocytes from patients with HD responded to PHA, even though the blood lymphocyte response was severely reduced. Lymph node lymphocyte response to PHA from patients with HD was variable, but correlated roughly with the blood lymphocyte response.It is hypothesized from the data presented that in HD, circulating thymus-dependent (T-)lymphocytes are stimulated by the presence of active disease. This stimulation of T-lymphocytes leads to a circulating T-cell depletion and to an increase in the number of cells circulating that are active in DNA synthesis. The degree of impairment of cell-mediated immunity would then depend upon the degree of T-lymphocyte depletion.     

Lack of effect of exogenous recombinant interleukin-2 on trauma-induced abnormalities in lymphocyte proliferation

Injury impairs cell-mediated immune function by depressing mitogen-induced lymphocyte proliferation and decreasing interleukin-2 (IL-2) production. In this study, we examined the ability of exogenous, recombinant IL-2 to restore lymphocyte proliferation after trauma. Recombinant IL-2 was added to cultures of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells obtained from seven healthy volunteers and from 21 victims of accidental trauma. In comparison to the healthy group, lymphocyte proliferation was reduced approximately 20% in patients with minor and moderate injuries, and 50% in patients with severe injuries. The addition of recombinant IL-2 to PHA-stimulated lymphocytes from injured patients did not substantially improve cellular proliferation. These results suggest that the depressed response of lymphocytes to mitogen stimulation after trauma is not due to decreased IL-2 generation.     

The short lived suppressor cell assay and its relationship with mitogen responsiveness in normal individuals

The short lived suppressor cell(SLSC) assay is an indirect test for spontaneous suppressor cell activity based on the principle that some suppressor cells are short lived in culture. It measures the ratio (suppressor index) between 3H-thymidine incorporation by lymphocytes stimulated by concanavalin-A (Con-A) after 24 hours of preincubation (during which SLSC are lost) to that incorporated by cultures stimulated immediately (SLSC present). In autoimmune conditions decreased suppressor indices are reported but low responses to mitogen stimulation also occur. The aim of this study was to investigate whether low levels of mitogen responsiveness could lead per se to low suppressor indices. A simultaneous analysis of suppressor indices and mitogen responses to Con-A, PHA and PWM of peripheral blood mononuclear cells in normal individuals was performed. There was an inverse correlation between responses to Con-A or PHA and the suppressor indices, indicating that low mitogen responsiveness does not lead per se to decreased SLSC activity and furthermore that the activity of SLSC may modulate lymphocyte reactivity in vitro.     

Impaired nuclear responsiveness to cytoplasmic signals in lymphocytes from elderly humans with depressed proliferative responses.

Lymphocytes from many elderly individuals exhibit depressed proliferative responses to the plant phytohemagglutinin (PHA). We have previously reported that this proliferative defect is not due to a failure to generate a cytoplasmic activator of DNA replication (ADR). In the present study, we tested the DNA synthetic response of nuclei derived from aged cells to an exogenous source of ADR. We found that nuclei from aged lymphocytes exhibiting low PHA responses were impaired in their ability to synthesize DNA in responses to ADR, compared with nuclei from younger adult donors. In contrast, those aged cells maintaining intact PHA responses provided nuclei that were also unimpaired in their response to ADR. The relationship between PHA responsiveness of the intact cells and ADR responsiveness of nuclei derived from these was a linear one. These results suggest that the depressed cellular reactivity of aged lymphocytes to PHA (when seen) may be due to a failure of these nuclei to respond to cytoplasmic stimulatory signals induced by the mitogen.     

Differential Effects of Immunosuppressants on Lymphocyte Function

In vitro and in vivo parameters of T lymphocyte function were evaluated in guinea pigs following treatment with the "cycle-active" drugs, 6-mercaptopurine (6MP) and methotrexate, and the "non-cycle-active" alkylating agent, cyclophosphamide. Commencing at the time of sensitization to tuberculin protein, animals were treated with an 8 day course of one of the cytotoxic drugs. This regimen either reduced or abolished the cutaneous response to PPD. The two cycle-active drugs inhibited the in vitro lymphoproliferative response to PPD and suppressed the elaboration of migration inhibition factor (MIF) by lymph node cells. However, these agents did not reduce blood lymphocytes, deplete the cellularity of the thymic dependent areas of peripheral tissues, or alter the in vitro response of lymph node cells to the nonspecific mitogen PHA. In contrast, treatment with cyclophosphamide was associated with a reduction in peripheral blood and tissue lymphocytes and impaired responses to PHA by residual lymph node cells. In vitro proliferative responses to PPD were inhibited but the capacity of lymph node cells to elaborate MIF was not suppressed. In addition to their effects on antigen-reactive lymphocytes, all three drugs significantly reduced the number of macrophages in induced peritoneal exudates. With respect to immunosuppressive activities, results of these investigations suggest that the noncycle-active agents affect both intermitotic and dividing T lymphocytes without impairing certain intermitotic functions of residual cells. The cycle-active drugs have a more restricted toxicity limited to those T lymphocytes which have been stimulated to undergo active DNA synthesis by antigenic challenge.     

Effects of moxalactam and cefuroxime on mitogen-stimulated human mononuclear leukocytes.

The effect of moxalactam and cefuroxime on mitogen-stimulated human peripheral blood mononuclear leukocytes was studied. Mononuclear leukocytes, mitogen, and antibiotic were added to microtiter wells. Cells were cultured for 3 days, pulsed with tritiated thymidine, and then counted. Compared with control cell cultures, treated cultures showed phytohemagglutinin responsiveness to be depressed by the addition of moxalactam at concentrations of 25 to 200 micrograms/ml (P less than 0.001) and by cefuroxime at concentrations of 50 to 200 micrograms/ml (P less than 0.02 to P less than 0.01). The depressive effect on blastogenesis was less marked when concanavalin A was used. Unstimulated lymphocyte transformation responses were also depressed by both antibiotics at all concentrations (P less than 0.05). Preincubation of mononuclear leukocytes with antibiotic for 2 h, followed by washing and culturing in an antibiotic-free medium, did not depress transformation response. When antibiotic was added 24 h after mitogen, depression of response was insignificant. The data from this study suggest that two new beta-lactam antibiotics, at concentrations achievable in serum when used therapeutically, may have immunosuppressant effects. It remains to be established whether these effects are clinically important.     

Meal induced increase in serum triglycerides has no effect on lymphocyte response to mitogens

The effect of a standardized heavy meal on the lymphocyte transformations induced by phytohaemagglutinin (PHA), concanavalin A (Con A) and PPD tuberculin was studied. The meal significantly increased the serum triglycerides (P less than 0.01), while it had no effect on cholesterol or high density lipoprotein-cholesterol levels. The increase in serum triglycerides did not affect lymphocyte transformation induced by phytohaemagglutinin or concanavalin A in whole blood microcultures. A slight decrease was observed when lymphocytes were stimulated with one out of three concentrations of PPD tuberculin (P less than 0.05). However, there was no correlation with the increase of triglycerides and decrease in lymphocyte transformation. Our observation shows that physiological changes in serum triglycerides do not affect the capacity of lymphocytes to respond to mitogenic stimulation, and the whole blood micromethod for lymphocyte stimulation to screen the capacity of cell-mediated immunity does not depend on the meal schedule of the patients.     

Lymphocyte reactivity to mitogens and tumor sex steroid receptors in breast cancer patients

In 60 breast cancer patients in stages I and II the blastogenic transformation of peripheral blood lymphocytes after phytohaemagglutinin (PHA) pokeweed mitogen (PWM) and concanavalin A (Con A) stimulation were assayed and estrogen (ER) and progesterone (PgR) receptor concentrations in tumor cytosol were measured. A negative correlation between lymphocyte reactivity to the mitogens and tumor steroid receptors concentration was found. The lymphocyte response to the mitogens in the patients with ER-PgR-tumors (R-) was significantly higher than in those with tumors either ER+PgR-or ER-PgR+ (R+) or ER+PgR+ (R++). There was also a negative correlation between lymphocyte response to PHA and either ER or PgR concentrations in the tumors. These results suggest that the presence of steroid receptors in tumor cells may be associated with the depression of immunological reactivity in breast cancer patients.     

SELECTIVE TRIGGERING OF HUMAN T AND B LYMPHOCYTES IN VITRO BY POLYCLONAL MITOGENS

Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.     

Comparison of ATP production in whole blood and lymphocyte proliferation in response to phytohemagglutinin

Lymphocyte proliferation in response to mitogens, phytohemagglutinin (PHA), concanavalin A, pokeweed, and/or specific antigens has been the method of choice for in vitro diagnosis of cell-mediated immune dysfunction. Recently, an assay to measure intracellular adenosine triphosphate (ATP) production in response to PHA has been developed that requires a shorter, overnight incubation. We compared a standard 5- to 7-day lymphocyte mitogen stimulation assay utilizing tritiated thymidine (3H-thy) incorporation to one in which ATP production in response to PHA by CD4-positive cells is measured in a luminometer that requires only 18-24 hr. A total of 20 patient samples suspected of having decreased cell-mediated immunity submitted for mitogen induced lymphocyte proliferation and 21 normal controls were tested in both assays. A comparison of these two methods has demonstrated that the screening ATP assay has a sensitivity at 24 hr of 100% in detecting decreased PHA induced lymphocyte proliferation at 5 days and a specificity of 85% in the samples obtained from normal controls. The data indicate that the ATP assay may be a useful screening tool for more rapid detection of blood samples with decreased cell-mediated immune responses. However, a positive screen should always be confirmed by 3H-thy uptake using mitogens and recall antigens like candida and tetanus.     

Inhibition of in vitro plaque formation by large granular lymphocyte leukemia cells from F344 rats

The effect of large granular lymphocyte leukemia on B lymphocyte function was studied by determining the number of plaques formed in an in vitro hemolytic plaque assay. Leukemia cells inhibited plaque formation by normal splenic lymphocytes in a logarithmic, dose-dependent manner. At the highest leukemia cell concentrations, spleen cell suspensions made 50% fewer plaques. Plaque forming responses were very sensitive to duration of preincubation time in all assays. The number of plaques formed decreased markedly if incubated 2 hr before the assay was performed. Incubation of the cells at 56 degrees C for 8 min did not alter the inhibitory activity but pretreatment with 0.01% trypsin did. Supernatant fluids from leukemia cell suspensions did not inhibit plaque formation. These data suggest that diffuse infiltration of lymphoid tissues by leukemia cells may interfere with some normal lymphocyte functions. Although leukemia cells inhibited splenic B lymphocyte function, leukemic rats did not have hypogammaglobulinemia.     

In vitro immunosuppressive effect: a comparative study between deoxymethylspergualin and cyclosporin A, on human lymphocyte responses

The immunosuppressive mechanism of Deoxymethylspergualin (MeDSG) on human lymphocyte responses in vitro was assessed in comparison with Cyclosporin A (CyA). Peripheral blood mononuclear cells from normal human volunteers were used for assay of mixed lymphocyte reaction (MLR), cell-mediated lympholysis (CML) and blastogenesis by PHA and OKT3. MeDSG suppressed only allogenic stimulation (MLR and CML), however, CyA showed a suppressive effect for all assays. Kinetic study of MLR showed that the suppressive activity did not decrease even when MeDSG was added at day 3 or day 4. CyA, however, showed a weak suppressive effect when added at a later phase of MLR. The results suggest that MeDSG is a useful immunosuppressive agent and, in vitro, its immunosuppressive mechanism on human lymphocyte responses is quite different from CyA.     

Studies of Cell Subpopulations Mediating Mitogen Hyporesponsiveness in Patients with Hodgkin''s Disease

Hodgkin's disease (HD) is associated with a deficit in T-cell immunity characterized by skin test anergy and decreased lymphocyte responses to phytohemagglutinin (PHA). To investigate this mitogen hyporesponsiveness in HD, we separated peripheral blood mononuclear cells on Ficoll-Hypaque gradients and determined their response to various suboptimal concentrations of PHA. As was expected, patients with HD demonstrated marked mitogen hyporesponsiveness relative to normal controls; however, if the cell suspensions were first passed through glass wool columns to remove adherent cells, the PHA responsiveness of the hyporesponsive HD cells was markedly increased. In contrast, the responsiveness of normal controls was decreased so that the responses of nonadherent normal and HD cells were statistically indistinguishable. Evidently, a glass wool-adherent suppressor cell had been removed from patients with HD, while a glass wool-adherent cell which enhanced mitogenic responses had been removed from normal controls during column passage. Previous to column depletion, patients with HD had decreased proportions of E-rosettes and increased proportions of cells with surface alpha-fetoprotein; however, the proportion of these cells was not changed after column passage. Significant changes with column depletion of glass wool-adherent cells in HD were recorded in the proportions of monocytes (13.2 vs 5.8%) and lymphocytes with C-3 receptors (12.6 vs. 7.8%). The only significant change in normal controls was a decrease in the proportion of monocytes (10 vs. 1.7%). To determine if glass-adherent cells would have a suppressor effect, HD-adherent cells were added in progressively increasing numbers to mononuclear cell suspensions depleted of glass wool-adherent cells. PHA responsiveness returned toward predepletion levels. In summary, patients with HD possess a glass wool-adherent suppressor cell which is responsible at least in part for in vitro mitogen hyporesponsiveness.     

Cellular Immunity to Nuclear Antigens in Systemic Lupus Erythematosus

Cellular immune repsonses were determined by skin testing and mitogen- and antigen-induced blastic transformation of peripheral blood lymphocyte cultures in 24 patients with systemic lupus erythematosus (SLE) and 24 normal subjects. The incidence of positive skin tests with Candida albicans, PPD (tuberculin-purified protein derivative) intermediate strength, Trichophyton and histoplasmin was not significantly different in the two groups nor was lymphocyte stimulation by the mitogen phytohemagglutinin-M (PHA-M), implying that cellular immunity is normal in SLE. However, the SLE patients had a significantly increased incidence of positive skin tests and stimulated lymphocyte cultures to a number of nuclear antigens compared with normal subjects. No correlation could be made between the test results and the activity of the SLE at the time of study except for a significant association between lymphocyte culture stimulation by rabbit thymus native DNA and active SLE nephritis. Patients with a membranous antinuclear factor (ANF) pattern had positive skin tests with rabbit thymus native DNA and usually had active disease.     

SPECIFIC ADHERENCE OF IN VITRO DIFFERENTIATED LYMPHOCYTES TO TARGET CELLS

Blast cells which were derived from rat lymphocytes by stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM) transformed within 2–3 days into a new type of lymphocytes when plated without mitogen on embryo fibroblast monolayers. These lymphocytes were termed secondary lyrophocytes. Upon addition of PWM to PWM-secondary lymphocytes a marked adherence to fibroblast monolayers was observed. The degree of adherence was estimated (a) by direct count of the lymphocytes in the medium and in the trypsinized fibroblast fraction, and (b) by using ⁵¹Cr-labeled lymphocytes. The adherence process required incubation at 37°C. The process started immediately after the addition of PWM and reached a plateau at 6 hr. At this time more than 80% of the lymphocytes adhered. In the absence of PWM only 12% of the lymphocytes were found in the fibroblast fraction. Unlike PWM-lymphocytes. Con A-lymphocytes, PHA-lymphocytes, and ordinary lymphocytes taken directly from the rat lymph nodes adhered only slightly more in the presence of PWM (10–20% adherence of ordinary lymphocytes) than in its absence (8% adherence). The adherence of the secondary lymphocytes and the ordinary lymphocytes was also studied in the presence of Con A and PHA. These mitogens induced high rate of adherence and they did not demonstrate specificity in their action. The adherence was accompanied by transformation of the lymphocytes to blast cells endowed with target-cell lytic ability. This transformation occurred mostly in the adhering fraction of the lymphocyte population. The results support the notion that target-cell recognition and destruction in cellular immunity involve contact between the cells.     

Inhibition of PHA-induced lymphocyte stimulation by factors in maternal serum during late pregnancy.

Immunosuppressive effect of pregnant serum obtained in the second and third trimesters was demonstrated in the PHA-induced homologous lymphocyte stimulation system, where 3H-thymidine incorporation into DNA was measured. Such an inhibitory factor was not present in the first trimester serum. The immunosuppressive principle detected in the second trimester serum was characterized by a potent inhibitory activity even at an increased concentration of PHA up to 75 micrograms, whereas the inhibitory activity found in the third trimester serum at optimum PHA concentration (15 micrograms) was not detectable when the PHA concentration was raised to 75 micrograms. Thus, the second and third trimester sera must contain different immunosuppressive principles.     

Expression and release of IL-2 receptor and production of IL-2 by activated T lymphocyte subsets.

T lymphocyte subsets differ in expression of cell surface antigen and functional properties. Both CD4+ and CD8+ subsets express interleukin-2 receptor (IL-2R) following their activation in vitro. In the present investigation T lymphocyte subsets were activated by different mitogens and IL-2R expression was enumerated on these stimulated subsets. Peripheral blood mononucleated cells (PBMC) were stimulated with phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) and then stained with anti-CD4 or anti-CD8 antibodies conjugated with fluorescein isothiocyanate and anti-IL-2R monoclonal antibody conjugated with phycoerythrin using a direct immunofluorescence technique. The percentage of IL-2R positive lymphocytes was enumerated by flow cytometry. The results showed that mitogen activated lymphocytes expressed variable degrees of IL-2R which were significantly higher than the control. 53% of CD4+ lymphocytes and 28% of CD8+ expressed IL-2R following PHA stimulation in vitro. Similarly, 47% of CD4+ lymphocytes and 23% of CD8+ lymphocytes expressed IL-2R following PWM stimulation. The present study also revealed that the release of soluble IL-2R (sIL-2R) and IL-2 production in supernatant from cultured PBMC varied with different mitogen stimulation. Using the same concentration of PHA and PWM as used to study IL-2R expression, higher activity of sIL-2R was detected in PHA stimulated lymphocytes as compared to PWM treated lymphocytes. However, IL-2 production was more in culture medium from PMW treated PBMC. Thus, there was a significant correlation between the cellular and soluble IL-2R but the production of IL-2 from activated PBMC cells had no good correlation with either the cellular IL-2R expression or the release of sIL-2R.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of human lymphocyte mitogen responsiveness and interleukin-2 production by polymorphonuclear leukocytes

The response of human peripheral blood lymphocytes to the mitogenic lectins phytohemagglutinin (PHA) and pokeweed mitogen (PWM) was examined in the presence of autologous polymorphonuclear leukocytes (PMN). Experiments were performed at sub-optimal and optimal mitogen concentrations employing lymphocyte: PMN ratios over a three log cell concentration range. Increases of up to 25,000-fold in mitogen stimulated lymphocyte proliferation as determined by 3H-thymidine incorporation were observed in PMN supplemented lymphocyte cultures as compared to lymphocytes cultured in the absence of PMN or with irradiated lymphocytes serving as filler cells. Similar results were obtained for PHA stimulated IL-2 production. The degree of enhancement of lymphocyte reactivity by PMN was also shown to be dependent on the source of serum supplementation (autologous versus xenogeneic). These results indicate that cell ratio is a critical factor in examining lymphocyte-PMN interactions as well as serum supplementation used. Early reports which have indicated a suppressive or no effect of PMN on lymphocyte reactivity based on a single lymphocyte: PMN cell ratio may need to be re-evaluated.     

THE DEVELOPMENT OF HYPERSENSITIVE LYMPHOCYTES IN CELL CULTURE

An in vitro cell-mediated immune response to pokeweed mitogen (PWM) is described. Rat lymphocytes were stimulated by PWM, by phytohemagglutinin (PHA), and by concanavalin A (ConA). In the presence of PWM only a fraction of the lymphocytes underwent blastogenesis. This was in contrast to the apparent total blastogenesis obtained in response to PHA or ConA. When blast cells derived from each of the mitogens were plated on rat fibroblast monolayer in the absence of mitogen they differentiated into a distinct type of lymphocyte termed "secondary lymphocyte." Addition of mitogens to cultures of these lymphocytes resulted in a retransformation to blast cells. The secondary lymphocytes were tested for their ability to effect lysis in the presence of each of the three mitogens. In. the presence of PWM, lysis of fibroblasts produced by PWM-lymphocytes was considerably more efficient than lysis obtained by ConA- or PHA-lymphocytes. No difference in effect on target fibroblasts was obtained when the three types of secondary lymphocytes were tested in the presence of either PHA or ConA. The stimulating action of PWM on lymphocytes was shown to be immunologically specific. No such specificity was found in the case of PHA or ConA. The results are interpreted to indicate that PWM combines with cell membranes and acts on the lymphocytes as a "transplantation antigen." Lymphocytes capable of responding to "PWM-transplantation antigen" transform to blast cells capable of specifically lysing PWM-conjugated fibroblasts. In the absence of the mitogen, PWM-induced blast cells differentiate to lymphocytes hypersensitive to PWM.