Cure of human melanoma lung metastases in nude mice with chronic indomethacin therapy combined with multiple rounds of IL-2: characteristics of killer cells generated in situ

We have shown that macrophage-derived prostaglandin (PG)E2 inactivates all interleukin 2 (IL-2) dependent killer cell lineages in the tumor-bearing host, so that chronic indomethacin therapy (CIT) combined with multiple rounds of IL-2 can cure experimental and spontaneous metastases of a variety of murine tumors. We tested the efficacy of this therapy on experimental human melanoma metastasis in nude mice and characterized the killer cells generated in situ. BALB/c nude mice were injected i.v. with 2 x 10(6) human lung-metastasizing line P52, MeWo melanoma cells. After 5 weeks, when lung nodules were well established, mice received vehicles alone (control) or were given (a) CIT (14 micrograms/ml in drinking water); (b) three rounds of IL-2, 25,000 Cetus U, 8 hourly i.p. (days 40-44, 50-54, 60-64); (c) CIT + three rounds of IL-2; (d) CIT + four rounds of IL-2 (round 4 on days 70-74); and (e) CIT + five rounds of IL-2 (round 5 on days 80-84). Control and experimental mice were killed on day 71 to score lung colonies and evaluate killer activity in splenic and lung lymphocytes and macrophages against murine YAC-1 lymphoma and B16F10 melanoma, human P52 melanoma, K562 erythroleukemia, and Raji lymphoma targets. Killer cells for P52 were phenotyped for Thy-1, Lyt-2, and asialo-GM-1 markers by ab + C'-mediated deletion of killer function. Mice in all groups were also kept for survival. CIT alone improved splenic NK activity but marginally reduced the lung colony counts or prolonged the survival time. Three rounds of IL-2 alone reduced the median colony counts by 50% and prolonged the survival by 2 weeks, but resulted in no long-term, disease-free survival, in spite of significant activation of LAK cells with Thy-1-, Lyt-2-, AGM-1+ phenotype in the spleen. CIT + 3 rounds of IL-2 reduced the median colony counts from 40 to 0 and improved the survival from a median of 66 (control) to 120 days (40% surviving 260 + days). CIT + four or five rounds of IL-2 caused long-term (260 + days) survival of 80% mice, most surviving 400 + days. The combination therapy activated killer lymphocytes (Thy-1-, Lyt-2-, AGM-1+) and, to a smaller extent, macrophages (AGM-1 +/-) in the spleen and the lungs, showing a high cytocidal ability for all the targets.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of histamine type-2 receptor antagonists on indomethacin and IL-2 immunotherapy of metastasis

Histamine type-2 receptor antagonists (H-2RA) have been used chronically to prevent dyspepsia in cancer patients subjected to immunotherapy with chronic indomethacin (Indo) and intermittent IL-2 in our cancer centre. We tested the effects of these agents during immunotherapy of C3H/HeJ mice transplanted s.c. with 5 × 10⁵ C3L5 mammary adenocarcinoma cells. Tumor-transplanted mice were divided into groups receiving: (1) Indo (14 µg/ml); (2) H-2RA, i.e. (a) ranitidine at 28.6 µg/ml (Ran-lo) or 143 µg/ml (Ran-hi), or (b) famotidine (Fam) at 4.3 µg/ml, or (c) cimetidine (Cim) at 107 µg/ml, all in the drinking water on days 5–24; (3) IL-2 (1.5 × 10³ Cetus U i.p. every 8 h on days 10–14 and 20–24); (4) combinations of H-2RA + Indo; or (5) combinations of H-2RA + Indo + IL-2. Animals were killed on day 24 for examination of primary s.c. tumor growth, secondary lung metastasis and splenocyte cytotoxicity against YAC-1 lymphoma cells (⁵¹Cr release assay). Results revealed: (1) primary tumor growth was reduced in mice treated with Fam + Indo, Indo + IL-2 and any of the H-2RA + Indo + IL-2 (no differences were observed within the last two groups); (2) lung metastases decreased in mice treated with IL-2 alone, Indo + IL-2, and Indo + IL-2 + Ran-hi; (3) splenic cytotoxicity was suppressed in tumor-bearing controls, with partial restoration seen in Ran (both doses), Ran-lo + Indo, Ran-lo + Indo + IL-2, and Cim + Indo + IL-2 treated groups. Nearly complete restoration was seen in Cim, Cim + Indo, Indo + IL-2, Ran-hi + Indo + IL-2, and Fam + Indo + IL-2 groups. Thus, addition of H-2RA did not alter the overall therapeutic efficacy of the standard Indo + IL-2 tumor immunotherapy.     

Establishment of Tac-negative,interleukin-2-dependent cytotoxic cell lines from large granular lymphocytes (LGL) of patients with expanded LGL populations

Cell lines were established from purified large granular lymphocites (LGL) isolated from the peripheral blood of seven patients with phenotypically homogeneous LGL expansions. LGL were stimulated with phytohemagglutinin (PHA) or recombinant interleukin-2 (rIL-2) and further expandedin vitro in IL-2-containing media. The surface phenotype of LGL, as assessed by monoclonal antibody staining, was T3⁺ T8⁺ in five patients, T3^– T8^– in one, and T3⁺ T8^– in another patient. The cells also expressed Leu 7, Leu 11, and/or OKM 1 markers in various proportions and were identifiable as LGL by their morphological and cytochemical features. The original surface phenotype of the unstimulated LGL was retained in the IL-2-dependent cell lines from each individual patient, i.e., T3⁺ T8⁺ cells originated T3⁺ T8⁺ cell lines and T3^– T8^– cells originated T3^– T8^– cell lines. Other markers, such as Leu 11 and OKM 1, were generally lost in culture. LGL proliferated in response to rIL-2 but did not express detectable IL-2 receptors, even after prolonged periods of culture. All cell lines from each individual patient had the same surface phenotype, and within the single lines, all of the cells expressed the same markers. Cell lines from two patients consistently displayed chromosomal abnormalities. Although different in the two patients, the abnormalities were identical in all of the lines from the same patient and detectable in most of the cells examined, suggesting a clonal origin for the abnormally expanded LGL populations. Freshly isolated LGL did not exert NK activity. However, the IL-2-dependent LGL lines acquired the ability to kill K562 target cells and to produce gamma interferon (-IFN). No direct correlation was observed between these two properties.     

Activation of nonselective cation channels in the basolateral membrane of rat distal colon crypt cells by prostaglandin E2

Ion channels in the basolateral membrane of colonic crypts were investigated with the patch-clamp technique during stimulation of secretion. Intact crypts were isolated from rat distal colon and the cell potential was recorded by addition of nystatin to the pipette solution. The cell resting potential in the base of the crypt was –74±1 mV (n=90). Addition of 100 M carbachol to the bath resulted in a transient hyperpolarization by 9 mV, which was probably due to the opening of basolateral K⁺ channels. In contrast, application of prostaglandin E₂ (PGE₂, 1 nM–1 M) caused a dose-dependent depolarization in the base of the crypt. With 1 M PGE₂ cells depolarized from -74±1 to –27±2 mV (n=26). Cell potential recordings in the midcrypt showed only a slight and transient depolarization after application of PGE₂, whereas cells close to the surface of the crypt had no response. In the base of the crypt the PGE₂-induced depolarization could be completely inhibited by addition of 50 M flufenamic acid, a known blocker of nonselective cation channels. After substitution of all monovalent cations by N-methyl-D-glucamine in the bath, PGE₂ had no significant effect on the cell potential. Cell-attached experiments with no nystatin in the patch pipette revealed the activation of ion channels in the basolateral membrane after application of PGE₂. After excision of the membrane patch, these channels could be identified as nonselective cation channels. Experiments involving substitution of the bath solution showed that the channel is impermeable for Cl^– and scarcely permeable for Ca²⁺ ions. The permeability sequence for monovalent cations, as calculated from reversal potentials, is NH ₄ ⁺ >Na⁺=K⁺>Rb⁺=Li⁺TRIS⁺=NMDG⁺. Single channels are completely inhibited by flufenamic acid (50 M), mefenamic acid (200 M), as well as by 3, 5-dichlorodiphenylamine-2-carboxylate. In conclusion, PGE₂ activates nonselective cation channels in the basolateral membrane of cells in the base of colonic crypts. It is suggested that this mechanism initiates the secretion of K⁺ ions. Na⁺ influx through the nonselective cation channel will stimulate the Na⁺/K⁺ pump and active uptake of K⁺ at the basolateral side. K⁺ can leave the cell at the luminal side through K⁺-selective channels.     

Effects of moderate endurance exercise and training on in vitro lymphocyte proliferation,interleukin-2 (IL-2) production,and IL-2 receptor expression

This study was designed to examine immunological responses to an acute bout of cycle ergometry exercise before and after moderate endurance training. Previously sedentary males were randomly assigned to matched training (n=9) or control (n=6) groups. Training comprised 12 weeks during which supervised cycle ergometer exercise took place [30 min at 65–70% of maximal oxygen intake , 4–5 days · week^–1]. An acute bout of exercise (60 min; 60% was performed initially and after the 12-week interval. Samples of peripheral venous blood were taken at rest, after 30 and 60 min of exercise, and at 30 and 120 min post-exercise. Training improved by an average of 20% (40.6 to 49.2 ml · kg^–1 · min^–1). Relative to baseline and control measures, the resting concentration of (CD3-CD16⁺/CD56⁺) natural killer (NK) cells increased by 22% (P<0.05). The resting count of total CD25⁺ [interleukin-2 receptor (IL-2R) chain] lymphocytes did not change following training, but dual staining analysis showed a 100% increase in the fraction of CD16⁺ CD25⁺ NK cells (P < 0.05). Likewise the resting CD122⁺ (IL-2R chain) lymphocyte count increased 35% after training, the greatest increases (44%) being in CD16⁺ CD122⁺ NK cells (P<0.05). Soluble IL-2R levels also increased 33% (P< 0.05) after training. Following acute exercise at the same relative intensity; trained individuals exhibited a larger increase in the NK cell count, reduced lymphocytopenia, and attenuation of exercise-induced suppression of lymphocyte proliferation and IL-2 production (P<0.05). In addition, smaller increases in CD4 and CD8 counts during exercise were noted, but with faster recovery post-exercise (P<0.05). Addition of recombinant IL-2 (rIL-2) to phytohemagglutinin-stimulated peripheral blood mononuclear cell cultures did not reverse exercise-induced suppression of cell proliferation, either before or after training. However, rIL-2 did augment the spontaneous blastogenesis of exercise and post-training samples relative to baseline (P < 0.05). We conclude that moderate endurance training is associated with sustained alterations in immune function, both at rest and when exercising. Further investigations are necessary to determine the impact on overall health and susceptibility to disease.     

Spontaneous production of interleukin 3 by T lymphocytes from autoimmune MRL/MP-lpr/lpr mice

MRL/MP-lpr/lpr (MRL/lpr) mice develop a lupus-like autoimmune disease and a massive generalized lymphadenopathy associated with proliferation of nonmalignant Thy-1⁺ Lyt-1⁺ cells. The mechanism(s) leading to outgrowth of these cells is unknown. We report here that Thy-1⁺, Lyt-1⁺, Lyt-2^? lymphocytes from spleens of MRL/lpr mice, but not from several strains of normal mice, spontaneously secrete IL3. The presence of IL3 is shown by: (a) the ability of the supernatants from unstimulated spleen cells of MRL/lpr (MRL/lpr SUP) to support growth of IL3 but not IL2 addicted cells and (b) the growth-promoting activity in MRL/lpr SUP was absorbed with IL3-dependent cells but not with IL2-dependent cells. Spontaneous release of IL3 was detected in supernatants from spleen cells of 6-week-old MRL/lpr mice and the titers of IL3 activity increased with age. Nylon wool-enriched cells from spleens of MRL/lpr mice proliferated in response to purified IL3 and IL3 secreted by MRL/lpr T cells, in a manner similar to nylon wool-passed cells from normal mice. The cells responding to both sources of IL3 were Thy-1⁺, Lyt-1⁺, Lyt-2^?. Thus, Thy-1⁺, Lyt-1⁺,2^? cells from spleen of MRL/lpr mice spontaneously secrete IL3 and respond normally to this lymphokine. Four Thy-1⁺, Lyt-1⁺,2^? cell lines derived from unstimulated spleen cells of MRL/lpr mice were established in culture with IL3. These IL3-sensitive T cell lines help syngeneic and H-2-compatible normal small “resting” B cells to mature into plasma cells secreting predominantly IgG₁, IgG₂ and IgA. Taken together, these data and previous findings that T cells from MRL/lpr mice have an impaired production of and response to IL2, strongly suggest that abnormal production of IL3 may account for the outgrowth of Thy-1⁺, Lyt-1⁺,2^? cells in the MRL/lpr mouse. Finally, a mechanism linking abnormal production of IL3 and B cell hyperactivity in these animals is proposed.     

Lyt phenotypes of primary cytotoxic T cells generated across the A and E region of the H-2 complex

Six different cell-mediated lympholysis (CML) combinations were established, four of which generated effector cells against the I-E, and two against the I-A molecule. The cell surface phenotype of effector cells was then determined by depletion of cytotoxic T lymphocyte (CTL) activity with antisera and rabbit complement (C) treatment. Both types of effector cells were completely eliminated by treatment with anti-Thy-1.2 antiserum plus C. Anti-Lyt-1.2 and C depleted anti-A and anti-E killer activity but did not eliminate CTL generated across a whole H-2 difference. One out of three different batches of anti-Lyt-2.2 antiserum did not deplete anti-A killer activity, while it efficiently eliminated CTL generated across the E region or whole H-2 difference. However, two batches of anti-Lyt-2.2 antiserum depleted also anti-A CTL activity. A quantitative difference between anti-A and anti-E CTL in terms of Lyt-2 expression was demonstrated by significant differences in recovery of killer activity, after treatment of these two types of CTL with a wide concentration range of the same anti-Lyt-2.2 antiserum and C. Thus it is concluded that anti-A killer cells have the cell surface phenotype of Thy-1⁺, Lyt-1, Lyt-2↑, whereas anti-E CTL are Thy-1⁺, Lyt-1↑, Lyt-2←. The data are discussed in the context of a possible association of Lyt phenotypes of T cells with the type of MHC antigens they recognize.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with (1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; (2) Mococlonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and (3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: (1) the majority of cortical thymocytes are strongly Thy-1⁺ (positive), strongly T-200⁺, variable in Lyt-1 expression, and strongly Lyt-2⁺; (2) the majority of medullary thymocytes are weakly Thy-1⁺, strongly T-200⁺, strongly Lyt-1⁺, and Lyt-2^? (negative); (3) a minority of medullary cells are weakly Thy-1⁺, T-200⁺, strongly Lyt-1⁺, and strongly Lyt-2⁺; (4) a small subpopulation of subcapsular lymphoblasts is Thy-1⁺, T-200⁺, and negative for the expression of Lyt-1 and Lyt-2 antigens; (5) a small subpopulation of subcapsular lymphoblasts is only Thy-1⁺ but T-200^? and Lyt^?; and (6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.     

Electrophysiological studies in principal cells of rat cortical collecting tubules ADH increases the apical membrane Na+-conductance

The mechanism of ion transport across principal cells of rat cortical collecting tubules (CCT) and its regulation by vasopressin (ADH) has been studied in the isolated perfused tubule. To amplify the response to ADH rats were treated with 5 mg I. M. desoxycorticosterone 4–9 days prior to the experiments. Addition of 2·10^–10 mol·1^–1 ADH increased the transepithelial voltage from –5.1 ±0.7 mV to –16.1±1.4 mV (n=37) and decreased the transepithelial resistance from 51±4 cm² to 39±2 cm² (n=33). Optical and functional differentiation of impalements of principal and intercalated cells was made and only data of principal cells are presented. ADH depolarized the apical membrane from 79±1 mV to 66±2 mV (n=26) and decreased the fractional resistance of the apical membrane from 0.76±0.04 to 0.70±0.04 (n=13). These ADH effects were prevented by 10^–5 or 10^–4 mol·1^–1 luminal amiloride which hyperpolarized the apical membrane when added in the presence or absence of ADH. Apical and basolateral membranes were dominated by large K⁺ conductances and addition of 3 mmol·1^–1 barium to bath or lumen perfusates increased transepithelial resistance almost two-fold, whereas luminal amiloride increased the transepithelial resistance only by 26–35%. Ouabain (0.5 mmol·1^–1, bath) depolarized the basolateral membrane and decreased its K⁺ conductance. These effects were prevented by the simultaneous presence of apical amiloride suggesting that the only route of Na⁺ entry into the principal cells occurred via the amiloride sensitive Na⁺ conductance. We conclude that ADH stimulates Na⁺ reabsorption and K⁺ secretion in the rat CCT primarily by increasing the Na⁺ conductance in the apical cell membrane.Parts of this study have been presented at the 19th ASN meeting in Washington, DC, USA 1986     

Circulating leucocyte and lymphocyte subpopulations before and after intensive endurance exercise to exhaustion

Summary Seventeen healthy cyclists [age 20.8 (SD 4.8) years; body mass 68.3 (SD 7.7) kg; body fat, 11.4 (SD 2.6) %; height, 179.1 (SD 5.9) cm; 60.9 (SD 7.4) ml · kg^–1 · min^–1] conducted intensive endurance exercise to exhaustion (stress test, ST) on a cycle ergometer at 110% of their individual anaerobic threshold [Th_an,individua; exercise intensity, 3.97 (SD 0.6) W · kg^–1 ; duration, 23.9 (SD 8.3) min; maximal lactate concentration, 7.39 (SD 2.59) mmol · 1^–1]. The distribution of leucocyte subpopulations was measured flow cytometrically: before, immediately after (0), 5 (+5), 30 (+30) and 60 (+60) min after ST. The lymphocytes (0 min) and granulocytes (+60 min) were mainly responsible for the increase of leucocytes. Lymphocytes were significantly lower at +30 and + 60 min than before. CD3^–CD16/CD56⁺ (+480%) and CD8⁺-lymphocytes (+211%) increased at 0 min more than the other lymphocyte subpopulations (CD3+-cells, +100%; CD4⁺ cells, +56%; CD19⁺-cells, +64%). CD3^–CD16/CD56⁺-and CD8⁺-cells also were mainly responsible for the decreased values of lymphocytes at +30 min and +60 min compared to before. At 0 min naive CD8⁺ cells (CD45RA⁺, CD45RO^–) increased more than memory CD8⁺-cells (CD45RA^–, CD45RO⁺). Changes of naive and memory CD4⁺-cells did not differ. All lymphocyte subpopulations, in particular CD8⁺- and CD3^–CD16/CD56⁺-cells, decreased rapidly between 0 min and 5 min. We conclude that an intensive endurance exercise to exhaustion causes a mobilisation of lymphocytes, especially of natural killer cells (CD3^–CD16/CD56⁺) and naive, unprimed CD8+ cells (CD45RA⁺, CD45RO^–) which may be transported to injured muscles. The decreased cell numbers of the latter subpopulations are possibly one reason for the susceptibility to infections during the first hours after exercise. Furthermore, an exact definition of the intensity of exercise and times of taking blood is essential for comparing results describing cell parameters during or after exercise.     

Dysregulated expression of the IL-2 receptor {beta}-chain abrogates development of NK cells and Thy-1+ dendritic epidermal cells in transgenic mice

The IL-2 receptor ß-chain (IL-2Rß), a specificity-determiningsubunlt In the IL-2R complex with a restricted tissue distributionpattern, Is essential for signal transductlon. Our previousstudies demonstrate that the continuous treatment of mice withanti-IL-2Rß) resulted in the complete disappearanceof NK cells and Thy-1⁺ dendritic epidermal cells (Thy-1⁺ dEC),suggesting that signals through IL-2Rß are criticallyinvolved in development of these lymphocyte subsets. However,these lymphocyte subsets are reported to be apparently unaffectedIn the IL-2-deficient mice. To further examine the biologicalroles of the IL-2Rß, transgenic mice carrying theIL-2Rß transgene were generated. In these mice, highlevels of the cell surface expression of the IL-2Rßwere observed in essentially all hematopoietic lineage cells,and CD4⁺ T cells as well as CD8⁺ T cells showed vigorous cellproliferation upon IL-2 stimulation. Surprisingly, NK cellsmarked with a high expression of NK1.1 in the spleen and Thy-1⁺dEC in the skin were completely absent in transgenic mice. However,the development of other lymphocyte subsets Including conventionalßTCR ⁺ cells, TCR⁺ cells and B cells remained apparentlyintact. From these observations together with previous dataon IL-2-deficlent mice, we speculate that factors, other thanIL-2 that utilizes the IL-2Rß as its functional receptorsubunlt, may have a vital role in the development of NK cellsand Thy-1⁺ dEC. Implications for possible In vivo functionsof over-expressed IL-2Rß are discussed.     

A Comparative Analysis of Cell Surface Markers on Murine NK Cells and CTL Target-Effector Conjugates

The rosetting of sheep erythrocytes (SRBC) coated with non-haemagglutinating monoclonal antibodies rather than conventional haemagglutinating antisera revealed readily detectable FcR on most splenic natural killer (NK) cells since 76% of splenic lymphocytes forming conjugates with YAC also resetted with SRBC coated with high concentrations of monoclonal anti-SRBC antibody of the IgG2b subclass and since Ficoll depletion or enrichment of splenic lymphocytes rosetting with IgG2b-coated SRBC resulted in a corresponding 4-fold decrease or increase in conjugate-forming cells and a 10-fold decrease or increase in NK cytolytic acttvity. NK cells bound much less readily to monoclonal IgG2a and not at all to monoclonal IgGI or IgM, but the degree of binding was directly proportional to the amount of antibody on the erythrocytes and was not isotype-restricted. In addition, immunofluorescent studies revealed that YAC-1-conjugated lymphocytes were Lyt-1^-, Lyt-2^-, partially Thy-1⁺ (60%), asiato-GMI ⁺ (80%), Qa-4⁺ (77%), Qa-5⁺ (79%), and Ly-5⁺ (94%). In comparison, a proportion (39%) of alloimmune peritoneal exudate cells which conjugated with P815–2 also siained by immunofluorescence with anti-asialo GM1 antisera. Most (>90%) P815- conjugated cells were Thy-1⁺, Lyt-2⁺. and a subpopulation of Lyt-l⁺2⁺ conjugates was observed (25 %). Qa-5 and Ly-5 were also expressed on most (two-thirds) cytolytic T lymphocytes (CTL) conjugates, whereas Qa-4 and FcR for IgG2b were not detected. The best phenotypic distinctions between NK cells and CTL were therefore based on the presence or absence of Lyt-2, Qa-4, and FcR for IgG2b on most effector cells. Anti-asialo-GMl or monoclonal anti-Qa-4 and complement treatment greatly diminished both the frequency of NK conjugates and the percentage of conjugates with detectable IgG2b FcR or asialo-GM1. These results confirm that NK cells co-express asialo-GMI and Fc receptors, at the single-celt level, and provide a simple method for greatly enriching NK populations at least 10-fold.     

P-glycoprotein-mediated multidrug resistance and lymphokine-activated killer cell susceptibility in ovarian carcinoma

The sensitivity of tumor cells to lysis by natural killer (NK) and interleukin-2 (IL-2)-activated killer (LAK) cells was studied in three ovarian carcinoma cell lines (2780.9S, SKOV-3, and CHOAUXB1), four multidrug-resistant (MDR) variants, and a melphalan-resistant line. The antitumor activity of LAK cells was evaluated both by⁵¹Cr release and by conjugate formation assays. Four of four P-glycoprotein-positive (P-gp⁺) MDR ovarian carcinoma cell line variants were lysed by human LAK cells to a greater extent than were their drug-sensitive counterparts. In contrast, a melphalan-resistant ovarian carcinoma cell line that does not overexpress P-gp (P-gp^–) did not exhibit an increased susceptibility to LAK cells relative to its parental cell line. Two of the four P-gp⁺ MDR ovarian carcinoma cell line variants were tested for human NK cell susceptibility and this was found to be unchanged or decreased. The P-gp⁺ MDR ovarian carcinoma cell line 2780.AD645 showed a higher frequency of tumor cell binding to LAK cells than did the drug-sensitive parental line. A monoclonal antibody (mAb) against a cell surface epitope of P-gp, MRK16, used at 1 g/ml, enhanced the LAK susceptibility of P-gp⁺ MDR ovarian carcinoma cell lines. However, when incubation with 10 g/ml MRK-16 antibody (Ab) was followed by 12.5 g/ml F(ab)₂ goat anti-mouse (GAM) immunoglobulin (Ig), the increased LAK susceptibility of P-gp⁺ MDR cell lines was inhibited. These data strongly suggest that P-glycoprotein-positive MDR ovarian carcinoma cells not only are targets for LAK cells, but are more sensitive than their drug-sensitive parental lines. This is in contrast to their susceptibility to NK cells, which is low to start with and remains unchanged or even decreased in MDR cells. It is postulated here that P-gp or associated changes result in a greater frequency of effector-target cell binding, leading to increased LAK cell cytotoxicity.     

Basolateral mechanisms of intracellular pH regulation in the colonic epithelial cell line HT29 clone 19A

The intracellular pH (pH_i) of the colonic tumour cell line HT29 cl.19A was studied by microspectrofluorometry using the pH-sensitive dye BCECF. Single cells within a confluent monolayer, grown in a polarized manner on permeable supports, were examined. An amiloride-sensitive Na⁺/H⁺ exchange and a stilbene-insensitive Cl^– /HCO₃ ^– exchange mechanism have been identified in the basolateral membrane. Removal of Na⁺ from the basolateral solution caused a decrease of pH_i by 0.50±0.09 unit (n=4). Amiloride or Na⁺-free solution at the apical side had no effect on pH_i. Cl^– removal at the basolateral side led to an increase of pH_i by 0.20±0.03 unit (n=4) whereas apical removal had no influence on pH_i. This effect was independent of Na⁺ and was insensitive to 0.2 mM 4,4-diisothiocyanatodihydrostilbene-2, 2-disulphonic acid. A basolateral Cl^–/ HCO₃ ^– exchanger is the most likely explanation for this observation. The Na⁺/H⁺ exchange mechanism in the basolateral membrane is an acid extruder, whereas the C1^–/HCO₃ ^– exchanger is an acid loader. Both of these mechanisms are important for the maintenance of intracellular pH in HT29 cl.19A cells.     

Age-related alterations of the lymphocyte population in the thymus of (NZB × SJL)F1 mice: Analysis by flow cytofluorometry

(NZB × SJL)F₁ (NS) female mice develop a marked hypertrophy of the thymus in the course of aging. The age-dependent evolution of the intrathymic lymphocyte population of these mice was monitored in comparison with that of the immunologically normal BALB/c and C57BL/6 strains. The expression of several lymphocyte markers on the surface of the thymic cells was quantitated using single- and two-color flow cytofluorometry analysis. Two main cell types could thus be identified in the thymus of 3-month-old mice: a major subset of bright Thy-1⁺, Lyt-1⁺2^?, bright peanut agglutinin (PNA)⁺ lymphocytes and a minor subset of dull Thy-1⁺, Lyt-1⁺2^?, dull PNA⁺ lymphocytes. In BALB/c and C57BL/6 mice, the frequencies of these cell types did not significantly vary between 3 and 18 months of age, despite a drop of thymic cellularity. In contrast, in NS female mice the proportion as well as the absolute number of dull Thy-1⁺, Lyt-1⁺2^?, dull PNA⁺ cells increased while those of bright Thy-1⁺, Lyt^?1⁺2^?, bright PNA⁺ cells diminished during the same period. The emergence of new cell types could also be noted in the thymus of aging NS female mice. Thus, in addition to the appearance of dull PNA⁺, Lyt-2⁺ cells and of bright PNA⁺ cells devoid of T cell markers, there was a high frequency of non-T cells bearing surface immunogloublin, Ia antigen and receptors for Fc. These data indicate that the thymic hypertrophy of NS female mice reflects the intrathymic accumulation of large numbers of phenotypically mature T and B cells. Such alterations were not detectable in NS male mice.     

Na+-HCO3 − cotransporter and intracellular pH regulation in chicken enterocytes

The current studies examine the presence of the Na⁺-HCO₃ ^– cotransporter in chicken enterocytes and its role in cytosolic pH (pH_i) regulation. The pH-sensitive dye 2,7-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pH_i. Under resting conditions, pH_i was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO₃ ^–. Removal of external Na⁺ decreased pH_i and readdition of Na⁺ rapidly increased pH_i towards the control values. These Na⁺-dependent changes were greater in HCO ₃ ^– than in HEPES-buffered solutions. In HCO ₃ ^– - free solutions the Na⁺-dependent changes in pH_i were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4-diisothiocyanatostilbene disulphonic acid (H₂-DIDS). In the presence of HCO ₃ ^– , the Na⁺-induced changes in pH_i were sensitive to both EIPA and H₂-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na⁺ and HCO ₃ ^– were present. This pH_i recovery, which was EIPA resistant, and dependent on Na⁺ and HCO ₃ ^– , was inhibited by H₂-DIDS and occurred at equal rates in both Cl^–-containing and Cl^–-free solutions. Kinetic analysis of the rate of HCO ₃ ^– - and Na⁺- dependent pH_i recovery from an acid load as a function of the Na⁺ concentration revealed first-order kinetics with a Michaelis constant, K _m, of 11 mmol/l Na⁺. It is concluded that in HCO₃ ^/– buffered solutions both the Na⁺/H⁺ exchanger and the Na⁺-HCO₃ ^– cotransporter participate in setting the resting pH_i in isolated chicken enterocytes and help the recovery from acid loads.     

Effects of severe arterial hypocapnia on regional blood flow regulation,tissuePO2 and metabolism in the brain cortex of cats

The effect of a stepwise decrease inPaCO₂ from 3.9–1.6 kPa on rCBF, rCMRO₂, tissuePO₂ and concentrations of glucose, lactate, pyruvate, ATP, ADP, AMP and phosphocreatine in the brain cortex was studied in cats lightly anaesthetized with sodium pentobarbital. 1. Moderate lowering ofPaCO₂ to 2.5 kPa induced in all animals a homogeneous decrease of rCBF in corresponding areas of the right and left hemisphere. Mean rCBF fell from 129.2 to 103.1 ml · 100 g^–1 · min^–1, while rCMRO₂ remained unchanged (12.7–12.9 ml · 100 g^–1 · min^–1). The tissuePO₂ frequency histograms showed a shift to lower values without indicating the presence of brain tissue hypoxia. 2. Severe arterial hypocapnia (PaCO₂=1.6 kPa) caused an inhomogeneous blood flow reaction. Both further decreased as well as increased rCBF values were measured simultaneously in the brain cortex of individual animals (mean rCBF=97.6 ml · 100 g^–1 · min^–1). At the same time tissuePO₂ measurements and metabolite assays indicated the presence of pronounced brain tissue hypoxia. The tissue concentrations of lactate and pyruvate and the lactate/pyruvate ratio were significantly increased, while the phosphocreatine concentration was significantly reduced. In addition, rCMRO₂ decreased to 11.3 ml · 100 g^–1 · min^–1. The results provide conclusive evidence that severe arterial hypocapnia leads to an insufficient O₂ supply of the brain cortex, which in turn seems to counteract the influence of hypocapnia on cortical blood flow regulation.Preliminary reports of these investigations were presented at the International Symposium on Oxygen Transport to Tissue, July 9–11, 1980 in Budapest and at the Second International Symposium on Pathophysiology and Pharmacotherapy of Cerebrovascular Disorders, July 22–25, 1980 in Tübingen, FRG     

DAB389IL-2 recombinant fusion toxin effect on lymphocyte- and macrophage-producing cytokine subpopulation cells in experimentally induced demyelinating disease in mice

Context: We have reported previously that DAB₃₈₉IL-2 recombinant fusion toxin targets IL-2R bearing CD4⁺ cells, and suppresses demyelinating disease in acute (A) - and chronic (C) - experimental autoimmune encephalomyelitis (EAE) animal models of multiple sclerosis.

Objectives: The present study was undertaken to investigate the effect of DAB₃₈₉IL-2 treatment on various cytokine-secreting cell populations in A-EAE and C-EAE mice.

Materials and methods: The effects of DAB₃₈₉IL-2 at doses of 200-, 800-, or 1600 kU administered i.v. on days 11–13 and 15 post disease induction on the clinical score and cytokine-secreting cell populations were examined using flow cytometry.

Results: C-EAE mice treated with 1600 kU DAB₃₈₉IL-2, but not A-EAE mice treated with 800 kU had significantly reduced disease. The CD3⁺CD25⁺ sub-population in spleens and spinal cords of A-EAE mice treated with 800 kU DAB₃₈₉IL-2 was increased, whereas in C-EAE mice treated with 1600 kU this population was decreased. DAB₃₈₉IL-2 treatment reduced CD3⁺CD4⁺, CD3⁺CD8⁺, CD4⁺CD8⁺, CD3⁺IL-2⁺, CD3⁺IFN-γ⁺ and CD3⁺TNF-α⁺ T cell subpopulations in the spinal cord in A-EAE, and C-EAE mice on day 16. CD11b⁺ macrophages that were IL-2-, IFN-γ-, and TNF-α- positive were reduced in A-EAE mice. DAB₃₈₉IL-2 treatment reduced CD19⁺ B-cells positive for IL-2 or CD11b⁺ in the spinal cord in acute and chronic disease. DAB₃₈₉IL-2 treatment also reduced lymph node CD3⁺CD8⁺, CD4⁺CD8⁺, CD3⁺CD25⁺ populations on day 16, and lymph node CD3⁺IL-10⁺ and peripheral blood CD3⁺CD25⁺ populations on day 24.

Discussion and conclusion: Our study demonstrates that DAB₃₈₉IL-2 fusion toxin suppresses EAE in a dose-dependent manner, and alters inflammatory cell sub-populations during disease development.     

An analysis of the cellular requirements for the production of soluble interleukin-2 receptorsin vitro

Following activationin vitro, peripheral blood mononuclear cells (PBMC) express cell-associated interleukin-2 receptors (IL-2R) and also release soluble IL-2R into culture supernatants. The present studies were undertaken to define which normal cells were responsible for the release of soluble IL-2Rin vitro. Both cell-associated and soluble IL-2R were quantitatively measured with a sandwich enzyme-linked immunoassay employing two monoclonal antibodies. PBMC were separated into populations of surface immunoglobulin-negative cells (T cells and monocytes) and surface immunoglobulin-positive cells (B cells and monocytes), and the T-cell population was further separated into OKT4-positive (OKT4⁺) cells and OKT4-negative (OKT4^–) cells. Following activation with phytohemagglutinin, pokeweed mitogen, and the monoclonal antibody OKT3, large amounts of soluble IL-2R were released by PBMC, unseparated T cells, OKT4⁺ T cells, and OKT4^– T cells. The population containing B cells and monocytes made small but readily detectable amounts of soluble IL-2R when stimulated with these T-cell mitogens; likely the result of contaminating T cells in the population. However, when highly purified B cells were stimulated withStaphylococcus aureus Cowan and recombinant IL-2, they also released small amounts of soluble IL-2R. The release of soluble IL-2R by T cells appeared monocyte dependent when OKT3, but not phytohemagglutinin, was employed for activation, and monocytes themselves released no detectable IL-2R under the conditions employed. These studies define the cellular requirements for the release of soluble IL-2Rin vitro and demonstrate that such receptors are released by B cells, T cells, and both OKT4⁺ and OKT4^– T-cell subsets.     

Presence of luminal K+, a prerequisite for active NaCl transport in the cortical thick ascending limb of Henle's loop of rabbit kidney

Previous data from our laboratory have shown that active transport in the cortical thick ascending limb of Henle's loop (cTAL), as measured by the short circuit current (I_SC, A · cm^–2), requires the presence of Na⁺ and Cl^–. The data were compatible with the model of secondarily active Cl^– reabsorption involving the cotransport of Na⁺ and Cl^– across the luminal membrane. The data suggested, furthermore, that 1 Na⁺ and 2 Cl^– interact with the luminal carrier. In the present study it was tested whether this reabsorptive mechanism also requires the presence of luminal K⁺. Isolated cTAL segments (n=40) were perfused at high flow rates with a modified Ringer's solution. Removal of K⁺ from the lumen reduced I_SC significantly from 215 to 133 A·cm^–2. Addition of Ba²⁺ (10^–3 mol·l^–1) which blocks the K⁺ conductance of the luminal membrane, to the K⁺-containing lumen perfusate decreased I_SC significantly from 234 to 141 A·cm^–2. Combination of both manoeuvres: perfusion with a K⁺-free and Ba²⁺-containing solution almost abolished I_SC from a control of 237 to 56 A · cm^–2. The results are compatible with the view that in rabbit cTAL the luminal carrier interacts with all 3 ions, possibly 1 Na⁺, 2 Cl^–, and 1 K⁺. K⁺ recycles across the luminal membrane through its conductive pathway.This study was supported by Deutsche Forschungsgemeinschaft Gr. 460/5-6-2