Characteristics of lymphoid subpopulations isolated from healthy subjects and those suffering from nonspecific ulcerative colitis

Results of the studies of functional activity of lymphocyte subpopulations T+G, T-G, "O" subpopulations isolated peripheral blood of healthy persons and those with nonspecific ulcerative colitis (NUC) have been presented in this paper. The functional activity of the cells was assessed by means of local xenogeneic graft-vs-host reaction performed on CBA mice and by determination of nonspecific esterase enzyme. It has been established that analogous lymphocyte subpopulations influence the graft-vs-host reaction development in different ways depending on the condition of cell donor state: health/pathology. "O" subpopulations isolated from healthy persons hampered the graft-vs-host reaction development, and on the contrary stimulated it in patients with NUC. The assay of nonspecific esterase in lymphocytes showed that the least activity was noted in T+G and "O" cells in nonspecific ulcerative colitis.     

Functional heterogeneity among peritoneal macrophages: I. Effector cell activity of macrophages against syngeneic and xenogeneic tumor cells

Peritoneal exudate cells from immunized and nonimmunized animals were separated into subpopulations by centrifugation on discontinuous bovine serum albumin (BSA) density gradients. Cells in the several subpopulations were then tested for their cytostatic or cytotoxic activity against syngeneic and xenogeneic tumor cells. Nonimmune macrophages isolated at the 8 to 11% BSA interface were highly inhibitory to the growth of syngeneic and xenogeneic tumor cells during coculture for 24 to 48 hr. A second macrophage subpopulation of heavier density was not as effective in preventing tumor growth and frequently augmented it. Cytotoxic activity against (C58NT) D tumor cells could not be detected with macrophages or subpopulations of macrophages from immune as well as nonimmune animals, as determined by a 4-hr chromium release assay. The cytotoxic activity of the immune peritoneal exudate cells observed by this assay could be accounted for by the small percentage of lymphocytes present.     

Characterization of bovine mononuclear cell populations with natural cytolytic activity against bovine herpesvirus 1-infected cells

Freshly isolated or overnight cultured peripheral blood mononuclear cells from immune or nonimmune animals had natural cytolytic activity against bovine herpesvirus 1 (BHV-1)-infected tumor target cells. No lysis was demonstrated against tumor target cells alone. This natural cytolytic activity was present in mononuclear cells from the spleen, lymph node, and peripheral blood but little or no cytolytic activity was detected in bone marrow or thymus cells. When monoclonal antibodies and complement to deplete bovine mononuclear cell subpopulations from the nonadherent cells were used, results indicated the effector cell was not a T cell, B cell, or activated monocyte. From nonadherent populations separated on density gradients, it was determined that the effector cells were large, low density mononuclear cells. These results indicate the nonadherent effector cells mediating lysis of BHV-1-infected xenogeneic adherent target cells were large null lymphocytes and/or immature monocytes.     

Specificity of allogeneic and xenogeneic cell recognition in the fetal lamb.

Cells prepared from liver, thymus, and spleen of fetal lambs at different stages if gestation were confronted with allogeneic and xenogeneic cells in MLC. Specific elimination of the responding cells with BUdR and UV light together with a subsequent restimulation was used to study the specificity of the reaction. The response of fetal liver cells was not based on the existence of specifically recognizing cellular subpopulations; the response was concluded to be due either to stimulatory products released by the stimulating cells or to the multipotentiality of the responding cells. Specifically recognizing cells first appeared in the thymus at 58 days postconception and in the spleen at 70 days. In the response of sheep lymphocytes against allogeneic and xenogeneic (mouse, human) cells, a cross-reactivity occurred. Fetal lamb lymphocytes were also capable of recognizing intraspecies differences on the xenogeneic cells. This capacity developed simultaneously with the specific recognition of allogeneic cells. No clear difference was observed in the reactivity of fetal thymus cells and spleen cells when compared to that of adult peripheral blood lymphocytes. These findings indicate that immunologically specific recognition of foreign cells is created in the sheep during the early intrauterine development.     

Characterization and sorting of mouse cytotoxic macrophages by their light scattering properties

The light scattering properties of mouse activated macrophages were analyzed by flow cytometry. Peritoneal adherent cells from B. abortus treated animals were found to segregate into two subpopulations as a function of their forward angle and 90 degrees angle light scatter. The cell subpopulations were separated by automatic sorting. The strongly scattering ones contained an elevated proportion of large volume and acid phosphatase rich cells. Their nonspecific cytotoxic activity against tumor cells was more important than that of weakly light scattering cells. Thus, flow cytometry might be helpful to characterize and isolate cytotoxic macrophage populations.     

Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes. NUC18 is not histone H2B

Glucocorticoids stimulate apoptosis in rat thymocytes that is characterized by internucleosomal DNA degradation. We have previously identified an 18-kDa calcium-dependent nuclease whose activity is associated with this DNA degradation. The existence of this nuclease has been challenged by Alnemri and Litwack (1989) J. Biol. Chem. 264, 4104-4111, who suggest that the nuclease we observed was histone H2B. We report here a modified nuclease assay which uses [32P] DNA as a substrate that has enabled the purification and characterization of the 18-kDa nuclease (NUC18). Using Bio-Rex 70 chromatography in conjunction with this assay, we show that NUC18 can be separated from histone H2B. Enzymatically active NUC18, purified to apparent homogeneity, failed to react with two different anti-histone H2B antibodies. NUC18 was inactive in the absence of calcium and known inhibitors of apoptosis, i.e. zinc and aurintricarboxylic acid inhibit its activity. Although NUC18 activity was detected in nuclear extracts of thymocytes of both control and glucocorticoid-treated thymocytes, these activities were distinct. Gel filtration analysis revealed that NUC18 was present as a high molecular weight complex (greater than 100 kDa) in both groups of cells, whereas it also existed as a low molecular weight form in glucocorticoid-treated cells. Thus, NUC18 remains a candidate for the endonuclease responsible for the DNA degradation component of the apoptotic process.     

Lysosomal enzyme activities in susceptible and refractory strains of Biomphalaria glabrata during the course of infection with Schistosoma mansoni

The distribution and abundance of the lysosomal enzyme markers, acid phosphatase (AP), peroxidase (PO), and nonspecific esterase (NE), within circulating blood cells (hemocytes) were examined in a schistosome-susceptible (PR albino M-line) and a resistant (10-R2) strain of Biomphalaria glabrata during the course of infection with Schistosoma mansoni. The dynamics of serum (cell-free hemolymph) AP activities and total hemocyte numbers in infected snails also were investigated. Hemocyte subpopulations, as determined by these enzyme markers, responded differently to parasite infection between snail strains. Generally, the hemocyte subpopulations within PR albino snails remained largely unchanged, whereas the same subpopulations in 10-R2 snails fluctuated considerably. The distribution of AP in the hemocytes of 10-R2 snails decreased by 1 hr postexposure (PE) to the parasite and remained low through 12 hr before increasing to control values at 24 hr and 2 wk PE. In comparison, PO activity increased by 1 hr PE and peaked at 12 hr before dropping to 0 hr values by 2 wk PE. The NE activity exhibited still another pattern with the percentage of NE-positive cells decreasing from 0 to 12 hr PE followed by a recovery to 0-hr values by 24 hr. The abundance of these hemocyte enzymes followed a similar pattern to that of their distribution, although some differences were observed. Serum AP values varied little in PR albino snails except for a significant increase at 2 wk PE, indicating a possible response to tissue damage resulting from migrating daughter sporocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin-dependent cell-mediated cytotoxicity in hamsters bearing syngeneic tumors

Spleen cells from LSH hamsters inoculated with xenogeneic, allogeneic, or syngeneic (PARA-7) tumor cells were assayed for their ability to mediate direct cell-mediated cytotoxicity (DCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) in a 4-hr chromium release assay. Spleen cells from animals immune to xenogeneic or allogeneic cells demonstrated specific DCMC against homologous target cells in the absence of Con A and nonspecific LDCC against both homologous and heterologous target cells in the presence of Con A. Spleen cells from animals bearing syngeneic PARA-7 tumors (TBA) failed to express DCMC against homologous or heterologous target cells; however, significant lysis of all target cells occurred in the presence of Con A. LDCC was not detectable when nonsensitized spleen cells from normal animals were employed. The LDCC reaction was dependent on the concentration of Con A and the number of effector cells present in the reaction. The development of LDCC effector cells in the TBA appeared to parallel the development of both DCMC and LDCC effector cells in immune animals.     

Nonspecific cytotoxic effects of antigen-transformed lymphocytes. III. Relationship to specific cytotoxicity.

Human peripheral blood lymphocytes, cultured either with PPD or in one-way mixed leucocyte reactions, develop a nonspecific cytotoxic potential which can be expressed on autologous as well as on unrelated xenogeneic target cells. This nonspecific cytotoxicity develops at an early stage in the mixed leucocyte reaction, before specific cytotoxic effects are demonstrable, and decreases as specific cytotoxicity increases.     

Functional heterogeneity among peritoneal macrophages. II. Enzyme content of macrophage subpopulations.

Rabbit peritoneal exudate (PE) macrophages were separated into subpopulations on discontinuous density gradients of bovine serum albumin. Four such macrophage subpopulations, referred to as bands A, B, C, and D (from lightest to heaviest buoyant density), were examined for differences in enzyme content. With regard to three acid hydrolases—acid phosphatases, β-glucuronidase, and cathepsin D—cells in bands A and B had greater enzyme activity than cells in bands C and D. A similar distribution of activities was observed for acid p-nitrophenylphosphatase. Peroxidase activity was present only in band D. Lysozyme activity was greatest in band D cells and least in band A cells. Only small differences in cytochrome c oxidase activity were observed among the subpopulations. Arginase activity was found to be greater in cells from band A than cells in bands B, C, and D. Macrophage subpopulations derived from PE macrophages placed in tissue culture for 7 days and macrophage subpopulation cells cultured for 2 days showed differences in acid phosphatase content similar to those seen with freshly obtained subpopulations. These results extend previous work demonstrating heterogeneity among PE macrophages.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. II. Responsible factors for nonspecific destruction

Cell-free supernatants of thoracic duct lymphocyte cultures which were stimulated in vitro by horse serum on syngeneic fibroblast monolayers are demonstrated to be cytotoxic on syngeneic embryonic fibroblasts by means of a direct cell count using microtest plates. Experimental supernatants showed up to 100% suppression of fibroblast growth at $\text{1}\text{3}$ dilution and up to 96% suppression at $\text{1}\text{4}$ dilution as compared to the control supernatants. Evidence is presented indicating that lymphocytes cultured on mosaic monolayers, which were comprised of syngeneic and xenogeneic fibroblasts, were reacting both to xenogeneic cells and horse serum in the medium at the cellular level. A hapten-to-carrier type relationship is suggested between xenogeneic antigen and horse serum. Absence of horse serum in the test cultures using these lymphocytes resulted in the abrogation of nonspecific toxic activity of lymphocytes while the specific activity, though diminished, remained. This again indicates the difference in the mechanisms underlying the specific and nonspecific target cell destruction by T cells.     

An evaluation of the metabolic basis of aflatoxin B1 toxicity by using buffalo granulocytes and agranulocytes in vitro

This study was aimed at monitoring cytotoxic changes in buffalo leukocyte subpopulations exposed to aflatoxin B1 (AFB1), since no such information is available for this species. The effects of AFB1 on glutathione (GSH) S-transferase, Ca2+Mg2+ATPase and protein synthesis in leukocyte subpopulations, namely, mononuclear (MN) cells and polymorphonuclear (PMN) cells isolated from the blood of the domestic buffalo (Bos bubalis), were studied. The cells were separated by using Ficoll-Paque and incubated in the presence of AFB1. GSH S-transferase activity was found to increase in cells exposed to AFB1, but there was no difference in activity between MN and PMN cells. PMN cell ATPase activity increased after AFB1 treatment, whereas no such effect was observed in the MN cells, which showed higher basal levels of ATPase activity. In the presence of AFB1, all the cells showed significant decreases in 14C-leucine incorporation, but the MN cells showed higher 14C-leucine incorporation than the PMN cells. Nevertheless, both cell types were affected by AFB1 and participated in its detoxification. There was also an appreciable decrease in the release of myeloperoxidase by activated PMN cells in the presence of AFB1.     

Response of human fetal lymphocytes in xenogeneic mixed leukocyte culture: Phylogenetic and ontogenetic aspects

Cells from liver, thymus, and spleen of human fetuses at different stages of development were capable of a proliferation response against xenogeneic and allogeneic lymphocytes. The kinetics of fetal responses against rat lymphocytes were identical to those of fetal and adult responses against allogeneic cells. With all of the cell types studied, including adult lymphocytes, allogeneic responses were stronger than xenogeneic. Xenogeneic responses against lymphocytes from rat, mouse, or sheep were stronger than those against lymphocytes from rabbit, chicken, snake, or frog. These results are interpreted to indicate that recognition of foreign lymphocytes by human lymphocytes depends on the phylogenetic position of the species used as a source of stimulating cells. The degree of recognition decreases as the phylogenetic distance increases. Specific elimination of responding cells and restimulation with another cell population was used to study the specificity of proliferation responses against mouse and rat lymphocytes. Responses by prethymic liver cells from human fetuses were not due to the existence of specifically recognizing subpopulations. Thymus and spleen at 16 weeks' gestation contained specific subpopulations capable of differentiating between xenogeneic and allogeneic cells, as well as between xenogeneic cells with different intraspecies histocompatibility patterns. Generation of receptor diversity on T lymphocytes is discussed briefly in the light of these findings.     

Interleukin 2-activated human killer cells are derived from phenotypically heterogeneous precursors

When cultured with native or recombinant human interleukin 2 (IL 2), human peripheral blood non-adherent mononuclear cells (NAMNC) acquire the ability to lyse both NK-sensitive and NK-resistant tumor target cells. The development of these IL 2-activated killer (IAK) cells, also known as LAK, is observed in the absence of exogenous antigen or mitogen. This study describes the ability of various subpopulations of human peripheral blood NAMNC with defined surface phenotype to generate the IAK activity. Human NAMNC were separated into various subpopulations on the basis of the ability to bind monoclonal antibodies, activated with IL 2, and were examined for the cytolytic effect on various tumor target cells. Although CD16+ (Leu-11+) NK cells from NAMNC could become IAK cells when cultured with IL 2, removal of these cells from NAMNC had no effect on the latter's ability to generate the IAK effect. When CD16- NAMNC were separated into CD2+ E rosette-forming T cells (ERFC) and CD2- non-T (non-ERFC) subpopulations, both subpopulations generated the IAK activity. The ability of monoclonal antibody-defined subpopulations of T and non-T cells to generate IAK cells was then examined. Both CD4+ and CD8+ subsets isolated by either positive or negative selection generated the IAK activity. Similarly, CD20+ (B1+) B cells and CD20- non-T (null) cells developed into IAK cells when cultured with IL 2. In contrast, Leu-7+ T cells failed to generate the IAK activity. CD4+ and CD8+ subsets were additionally separated into narrower subpopulations by using monoclonal antibodies anti-Leu-8 and 9.3 respectively, and were examined for their ability to generate IAK cells. Precursors of IAK cells were derived from each of the four: CD4+, Leu-8+ (inducer), CD4+, Leu-8- (helper/amplifier), CD8+, 9.3+ (cytolytic), and CD8+, 9.3- (suppressor) subpopulations of T cells. Thus, the IAK activity appears to be derived from phenotypically heterogeneous and otherwise functionally diverse human lymphoid cells and is not confined to any single subpopulation.     

Antigen-specific and nonspecific mediators of T-cell/B-cell cooperation. VI. Distinct patterns of cross-reactivity by two human gamma-globulin-primed helper T-cell subpopulations.

We have previously characterized the activities, in vitro, of two different helper T-cell subpopulations, primed with human γ-globulin (HGG). One T-cell subpopulation helps the response of B cells to determinants (e.g., haptens) bound to the same antigen to which the T cells are primed (specific help); the other helper T-cell subpopulation responds to the same priming antigen by secreting a nonspecific molecule which helps B-cell responses to erythrocyte antigens co-cultured with the priming antigen (nonspecific help). These subpopulations also differ in their frequency and dose response to antigen, both in vivo and in vitro. They are similarly susceptible to the induction of unresponsiveness to HGG. In order to determine whether these T-cell subpopulations share or differ in their ranges of antigen recognition, we have compared the reaction of these two HGG-primed helper T-cell subpopulations to a number of γ-globulins (γG's) from other species. Plaque-forming cells generated in response to HGG shared little or no cross-reactivity with any of the heterologous (γG's) tested. In contrast, HGG-primed nonspecific helper T cells responded with significant cross-reactivity when challenged in vitro with dog γG, but HGG-primed specific helper T cells did not respond with any such cross-reactivity. No other heterologous γG tested stimulated any significant cross-reactivity from either HGG-primed T-cell subpopulation. Thus, these two T-cell subpopulations differ in their antigenic recognition. Possible explanations of these data include: (i) a difference in receptor specificity; (ii) a difference in the receptor affinity; (iii) a difference in Ia determinants of the two subpopulations.     

Isolation and characterization of cell cycle-enriched subpopulations of a murine macrophage cell line by centrifugal elutriation

The cell cycle of the P388D 1 murine macrophage line was delineated and suspensions of exponentially growing cells were separated by centrifugal elutriation into subpopulations enriched in the various phases of the cycle. Analysis of both growth and labelled mitoses curves disclosed that the doubling and cell-cycle times were essentially identical (18.4 and 18.3 h), indicating that all cells were in cycle. In addition, G1 + 1/2M was 4.3 h, whereas S phase and G2 + 1/2M lasted about 12 and 1.5 h. The most homogeneous subpopulations of phase-enriched cells obtained by elutriation were cells in G1 and S, where purities (estimated by both labelling indices and analyses of DNA histograms obtained by flow cytometry) exceeded 80%. Isolation of G2 + M-phase cells was not as efficient, although the purity of these subpopulations was consistently greater than of 50%, an approx. 10-fold enrichment over unseparated suspensions of cells. Comparison of IgG2a-Fc-receptor-mediated phagocytic activities among the phase-enriched subpopulations showed that cells in G2 had appreciably enhanced activity.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. I. Kinetics

An in vitro culture method giving a high cell recovery rate of 25–38% has been developed to generate a relatively pure population of T cell progeny reacting to xenogeneic cell antigen. The method is basically a modification of the Ginsburg method and utilizes mosaic monolayers that are comprised of syngeneic Lewis and xenogeneic BALB/c fibroblasts. Thoracic duct lymphocytes of Lewis rats were cultured for 6 days on these monolayers and the resulting lymphocyte population, rich in blast cells, was collected and separated free of contaminating fibroblasts by an additional 6 hr incubation without monolayers. Cytotoxic activity of these harvested lymphocytes was assessed by a modification of the Hellström method in which embryonic target fibroblasts, Lewis or BALB/c, were plated at a critical density of 2200 cells/cm² of culture area. This, coupled with the use of a newly developed culture medium, allowed the demonstration of specific as well as nonspecific activity of up to 100% suppression at 48 hr of test incubation. The specific activity was clarified and determined critically by comparing the activity of these lymphocytes to that of lymphocytes grown on syngeneic monolayers, though rich in blast cells, reacting only to the horse serum in the medium. The nonspecific activity was not due to the deterioration of test cultures and needed the continued presence of fully active blast cells for complete suppression of the target cell growth in the later phase of test cultures. By changing the ratio of lymphocytes to target cells and following the kinetics of target cell destruction, it was shown that specific activity effects an active killing of target cells, whereas the nonspecific activity was primarily the suppression of target cell growth. The specific activity was effective with far less lymphocytes per target cell; was nearly complete by 12 hr of test cultures while the nonspecific activity was complete at 48 hr. Yet, the increase in the specific activity was nearly proportional to that of nonspecific activity and even the nonspecific activity seemed to kill sensitive Lewis target cells at its full intensity. These observations are in accordance with an interpretation of the mechanism of the specific target cell destruction as involving contact and recognition followed by the release of lymphotoxin into or onto target cells.     

Human interferon-gamma receptor. Mapping of epitopes recognized by neutralizing antibodies using native and recombinant receptor proteins.

Monoclonal antibodies produced against native interferon-gamma receptor (IFN gamma-R) have been characterized for their capacity to react with purified receptor and receptor-positive cells, to inhibit the binding of IFN gamma to cellular receptor, to precipitate the receptor protein when cross-linked to IFN-gamma, and to recognize the recombinant interferon-gamma receptor and 19 overlapping fragments of this protein expressed in Escherichia coli. The results of this analysis showed that: (i) the extracellular portion of human IFN gamma-R is located between the N terminus and the transmembrane region (amino acids 18-246). (ii) The intracellular domain is between the transmembrane region and the C terminus (amino acids 269-489). (iii) The monoclonal antibodies that react with the IFN gamma-R intracellular domain recognize small linear epitopes. (iv) The human IFN gamma-R binding site is located between the N terminus and the transmembrane region. (v) The monoclonal antibodies that react with IFN gamma-R extracellular domain and inhibit the binding of IFN gamma recognize two different epitopes. One of these epitopes (included between amino acids 26 and 133) is very close to the binding site for IFN gamma. The second (included between amino acids 70 and 210) is related to the binding site for IFN gamma without including it. (vi) These two functional epitopes are conformational and need S-S bridges to maintain their architecture. (vii) These conformational epitopes are formed in receptor fragments expressed in E. coli.     

beta-D-N-acetylglucosaminidase, a new cytochemical marker of human lymphocyte subpopulations

beta-D-N-acetylglucosaminidase staining characteristics of rosetted or non-rosetted normal and malignant lymphoid cells were compared with those observed after nonspecific esterase and acid phosphatase staining. With the three cytochemical techniques a similar staining pattern was observed in T cells (E-rosettes), their subpopulations T mu and T gamma, B cells and the non-T, non-B cells, as well as in the T cell populations defined with the monoclonal antibodies OKT3,4 and 8. T mu cells mostly displayed a "dot-like" reaction, T gamma and the non-T, non-B cells a "fine to heavy granular" reaction, while most B cells were negative. OKT4 and OKT8 positive lymphocytes showed for the larger part a dot-like staining pattern, however, the frequency of cells with a granular pattern was distinctly higher in the OKT8, than in the OKT4 positive cells. E(+)mIg(-) and E(-)mIg(-) A.L.L. blasts stained either with a dot-like or granular pattern or failed to react when stained cytochemically for beta-D-N-acetylglucosaminidase, nonspecific esterase and acid phosphatase activity. Only in a few instances a discrepancy was observed between the types of staining for esterase and acid phosphatase on one hand and those for beta-D-N-acetylglucosaminidase on the other.     

Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: 1) cell volume; 2) cell cycle position; and 3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings 1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and 2) demonstrate EC subpopulations in culture.