Inhibition of leukocyte adherence by 3-M potassium chloride extracts of human colorectal tumors.

Leukocyte adherence inhibition (LAI) assays were performed to test whether peripheral blood leukocytes (PBL) from patients with colorectal cancer could be inhibited from attachment to a glass surface when tumor-associated antigens (TAA) of human colorectal tumors were present. The assays were performed with PBL from 41 patients with adenocarcinoma of the colon or rectum with 3-M KCl extracts of colorectal tumors. The results demonstrated the presence of colorectal TAA in the 3-M KCl extract of colorectal tumor materials. The reactivity of leukocytes from patients with a favorable prognosis showed an increasing LAI trend; the reactivity of leukocytes from patients with an unfavorable prognosis had a decreasing LAI trend. In individual patients, alterations in the sequential LAI results paralleled changes in the clinical status, which thus strongly indicated that the LAI assay could be of value in the assessment of antitumor immunity.

Absence of thymus-derived lymphocyte markers in myelogenous leukemia (Ph1+) cell line K-562.

The myelogenous leukemia cell line K-562 with a Ph1+chromosome, derived from a patient with chronic myelogenous leukemia in terminal blastic crisis, is not a bone marrow-derived lymphoblastic cell line, because the cells neither produce immunoglobulins nor possess complement receptors. Since it has been suspected that blasts found in some patients with chronic myelogenous leukemia in blastic crisis might be thymus-derived cells, we have studied several parameters to demonstrate that K-562 cells are not thymus-derived lymphoblasts. The results of this study show: (a) no cross-reactivity of antisera to K-562 cells with normal human thymocytes; (b) lack of cytotoxicity of a specific horse anti-human thymocyte globulin for K-562 cells; (c) failure of the treatment of K-562 cells with bovine thymosin to induce antigenic determinant and erythrocyte rosette receptors on K-562 cells; (d) presence of receptors for the Fc portion of immunoglobulin G; (e) absence of terminal deoxynucleotidyl transferase; and (f) cytotoxicity of monkey antiserum to K-562 cells for malignant thymus-derived cells (Molt-4). However, absorption with Molt-4 cells abolished the cross-reactivity with Molt-4 cells, whereas 60% of the antibody to K-562 cells remained in the immune serum. Studies of DNA polymerase activities revealed that K-562 cells have levels of polymerase alpha and beta, like other proliferating cells, and an RNA-dependent DNA polymerase activity, presumably representing polymerase gamma.

Effects of spaceflight on rat peripheral blood leukocytes and bone marrow progenitor cells.

The white blood cell (WBC) elements and the bone marrow myeloid progenitor cell populations were analyzed to ascertain adaptation to micro-gravity and subsequent readaptation to 1 G in rats flown on the 14-day Spacelab Life Sciences-2 (SLS-2) mission. Bone marrow cells were harvested from one group of rats killed inflight (FD13) and blood was drawn from three other groups at various times. The WBC level was normal on FD14 with the exception of neutrophilia. On FD13, numbers of colony-forming units-granulocyte (CFU-G), CFU-GM, and CFU-M from flight animals were decreased compared with ground controls when incubated with recombinant rat interleukin-3 (rrIL-3) alone or in combination with recombinant human erythropoietin (rhEpo). On recovery (R + 0), flight rats had decreased numbers of total leukocytes and absolute numbers of lymphocytes and monocytes with elevated neutrophils compared with control rats. They had lower numbers of CD4, CD8, CD2, CD3, and B cells in the peripheral blood but no differences in spleen lymphocytes.

Effects of spaceflight on the number of rat peripheral blood leukocytes and lymphocyte subsets.

Experiments were carried out on peripheral blood leukocytes and spleen lymphocytes from 29 male rats that were flown during the Spacelab Life Sciences 1 (SLS-1) nine-day mission on the shuttle Columbia in June 1991 and on appropriate ground controls. On the day of landing, there was a significant decrease in the total white blood cell counts (P < 0.0001) of flight animals in comparison to controls. There was also a significant decrease in the absolute number of lymphocytes (P < 0.0001) and monocytes (P < 0.0001) in the flight animals. A slight decrease in the absolute number of eosinophils and a slight increase in the number of neutrophils were observed at landing, compared with preflight values. Immunophenotyping of the peripheral blood and spleen lymphocytes of flight and control animals indicated that, on the day of landing, there was a decrease in the absolute number of CD4 and CD8 positive cells and B lymphocytes. However, relative percentages of peripheral blood CD4+, CD8+, and B cells were not found to be depressed. No differences were discerned in the percent reactivity of spleen lymphocytes of flight animals compared with controls. The observed decrease in the number of leukocytes and lymphocytes at the immediate postflight period was transient and all values returned to the control levels by nine days postflight.

Differential regulation of interleukin-1alpha and interleukin-1beta in K-562 cells.

Interleukin (IL)-1alpha and IL-1beta are encoded by two separate genes, but both function as comitogens for lymphocyte activation. In this study, we observed K-562 cells to express constitutively mRNA for IL-1alpha, although IL-1alpha was not detected in the growth-conditioned medium (GCM). However, IL-1beta mRNA was not expressed unless the cells had been treated with phorbol myristate acetate (PMA). Both IL-1alpha and IL-1beta were detected in the GCM after the cells had been cultured with PMA, suggesting that IL-1 elaboration required PMA treatment. The K-562 cells treated with PMA differentiated to the myeloblastic stage, as observed by nuclear morphologic properties by electron microscopy. PMA treatment induced de novo expression of CD61 or gpIIIa, a marker associated with megakaryoblasts. These results showed that although K-562 cells constitutively expressed IL-1alpha mRNA, PMA treatment was required for secretion. On the other hand, both the expression and secretion of IL-1beta required treatment with PMA. This study showed that K-562 cells treated with PMA differentiated to the myeloblastic stage and expressed and secreted IL-1alpha and IL-1beta.

Simultaneous flow cytometric measurement of K-562 megakaryocytic differentiation and CD56+ large granular lymphocyte cytotoxicity.

K-562 cells have the capacity to undergo multi-lineage differentiation, which may be crucial to their ability to serve as target reservoirs for CD56+ large granular lymphocytes (LGL). Conventional techniques using chromium release assays to measure lymphocyte-mediated cytotoxicity suffer from disadvantages, including radioactive contamination and the inability to simultaneously determine K-562 and/or CD56+ lymphocyte phenotypes. We illustrate here a three-color flow cytometric method providing for the simultaneous evaluation of K-562-CD56+ LGL binding, K-562 cell viability, and the status of K-562 cell differentiation. Phorbol 12-myristate 13-acetate (PMA) engenders megakaryocytic differentiation in K-562 cell populations, as measured by presentation of the beta(3) integrin (gpIIIa, CD61), while maintaining a negative expression of MHC-I and MHC-II molecules. Using the auto-fluorescence of K-562 cells, flow cytometry can be used to demonstrate a significant decrease in CD56+ LGL activity against K-562 cells in populations pre-incubated with PMA. The capacity of three-color flow cytometry to measure lymphocyte-target cell binding and cell death kinetics, while simultaneously determining target cell phenotype, permits the specific localization of CD61-expressing K-562 cells to areas inconsistent with CD56+ LGL-mediated patterns of lysis.

Inhibition of leukocyte adherence by a 3 M potassium chloride extracts of human malignant melanoma.

The tube leukocyte adherence inhibition (LAI) assay was performed using 3 M KCl extracts of malignant melanoma materials and leukocytes taken from 24 malignant melanoma patients. Of these patients, 21 participated in sequential studies which employed a minimum of 3 time points. An increasing non-adherence index (NAI) was found in 5 of 6 patients who exhibited a favorable disease course. In 5 of 6 patients with an unfavorable disease course, NAI values decreased. In 8 of 9 patients with stable disease, the NAI also was stable. These results indicate that the trend of sequential LAI studies reflects the clinical course of malignant melanoma patients.

Diversity of cell surface hematopoietic antigens on K-562 sublines identified with monoclonal antibodies.

The surface antigen profile of 8 sublines of K-562 cells, the original line, and the clone RA6 was determined with a panel of 12 monoclonal antibodies reactive with hematopoietic cell differentiation antigens. Cells from all sublines expressed the precursor hematopoietic antigen reactive with RFB-1, the T-cell antigen reactive with OKT17, the B cell/granulocyte antigen reactive with BA-1, the My-1 antigen, and glycophorin A which reacted with R10. A low percentage of cells in some of the sublines expressed platelet/monocyte glycoprotein I binding AN51, monocyte antigen binding 63D3, and an erythroblast/monocyte/platelet antigen binding 5F1. The use of a panel of K-562 sublines demonstrates that K-562 cells do share several "common" antigens but express a marked diversity and variability of other hematopoietic antigens.

Cell death in the human leukemia cell line, K-562, induced by antiserum and monoclonal antibodies.

Rabbit polyclonal antiserum, or derived gamma globulin, to K-562 cells induces decreased TdR uptake within hours and cell death without cytolysis in 2-4 days. A panel of nine mAb, reactive with K-562 cells, was grouped on the basis of no effect on growth or TdR uptake, increased uptake, or decreased uptake. Treatment of cells with antiserum, gamma globulin, or mAb of the last group caused single-strand, but not double-strand, DNA fragmentation at a time when the cells were still viable. Cycloheximide did not inhibit the antibody effect suggesting that protein synthesis was not required. Aurintricarboxylic acid at certain concentrations markedly enhanced TdR uptake and protected the cells when antiserum was used but did not protect from mAb treatment.

Continuous or modulation expression of hematopoietic cell antigens in sublines of the leukemia cell line, K-562.

K-562 is described as a pluripotent leukemic cell line which expresses a diversity of hematopoietic cell differentiation antigens. With the use of monoclonal antibodies (MoAb), we show that the expression of these antigens is either "modulated", i.e. they are not expressed in early logarithmic growth but are expressed in late logarithmic growth, or "continuous", i.e. they are not affected by proliferation and phase of culture growth. Whether an antigen is modulated or continuously expressed appears to be an inherent property of the subline, since the expression of myeloid antigen binding the MoAb, PM81, was modulated on cells from a clone, whereas, the expression of this antigen was continuous by the parent subline and an independent subline F-1. Continuously expressed antigens appear to be an integral component of the K-562 cell surface matrix, while modulated antigen expression appears to be influenced by culture growth conditions.

PMA-treated K-562 leukemia cells mediate a TH2-specific expansion of CD4+ T cells in vitro.

Highly enriched preparations of human CD3+CD4+ T-lymphocytes were stimulated with mitogen or OKT3 to determine the capacity of K-562 cells to function as accessory cells. Phorbol 12-myristate 13-acetate (PMA)-treated K-562 cells were induced to differentiate along the megakaryocytic lineage and could supplant monocyte-accessory cell function. Intracytoplasmic analysis of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) established that IL-4, and not IFN-gamma, was preferentially produced by the activated lymphocytes. This polarized stimulation is compatible with a type 2 or humoral immune response of purified T cells co-cultured with differentiated K-562 cells in vitro, and may have implications in immunoregulation due to disease progression.

Effects of spaceflight on rat erythroid parameters.

Hematologic studies were performed on 21 ground control rats and 21 rats flown during the Spacelab Life Sciences-2 14-day mission. Group A (n = 5) was used to collect blood in flight and 9 days postflight, group B (n = 5) was injected with recombinant human erythropoietin (rhEpo), group C (n = 5) received saline as a control, and group D (n = 6) was killed in flight and tissues were collected. Results indicated no significant changes in peripheral blood erythroid elements between flight and ground control rats. The nonadherent bone marrow on flight day 13 showed a lower number of recombinant rat interleukin-3 (rrIL-3)-responsive and rrIL-3 + rhEpo-responsive blast-forming unit erythroid (BFU-e) colonies in flight rats compared with ground control rats. On landing day, a slight increase in the number of rhEpo + rrIL-3-responsive BFU-e colonies of flight animals compared with ground control rats was evident. Nine days postflight, bone marrow from flight rats stimulated with rhEpo alone or with rhEpo + rrIL-3 showed an increase in the number of colony-forming unit erythroid colonies and a decrease in BFU-e colonies compared with ground control rats. This is the first time that animals were injected with rhEpo and subsequently blood and tissues were collected during the spaceflight to study the regulation of erythropoiesis in microgravity.

Lymphatic tissue changes in rats flown on Spacelab Life Sciences-2.

Thymus, spleen, inguinal lymph node, and bone marrow specimens from rats flown on the 14-day Spacelab Life Sciences-2 mission were examined after staining of tissue sections. The primary observation was a transient retrogressive change in lymphatic tissues in the rats within a few hours after landing. There was a diffuse increase in tingible body-containing macrophages in the cortex of the thymus, thymus-dependent areas of the splenic white pulp, and inguinal lymph node. This was not observed 9 days after recovery. The in situ labeling of fragmented DNA strands catalyzed by exogenous terminal deoxynucleotidyltransferase (TdT) with ApopTag reagents (Oncor, Gaithersburg, MD) inside the tingible body-containing macrophages indicated that the process was one of apoptosis. No increase in tingible body macrophage activity was noted in thymus and spleen tissue obtained from rats in flight on flight day 13. The reaction to gravitational stress from readaptation to 1 G is the most likely explanation of the transient retrogressive change in lymphatic tissues.

Effects of microgravity and increased gravity on bone marrow of rats.

Astronauts have a reduction in their red cell mass when exposed to microgravity. This is probably mainly due to a physiological response to decreased energy requirements. Further studies of erythropoiesis were carried out in microgravity on rats flown on Soviet Biosatellite 2044 and in hypergravity by centrifugation at 2G. Studies included: bone marrow cell differential counts, clonal studies of RBC colony formation, and plasma erythropoietin determinations. In the bone marrow of Cosmos flight animals there was a slight increase in granulocytic cells and in centrifuged animals, a slight decrease in the percentage of erythroid cells which led to an increased M:E ratio. The bone marrow cells of flight and centrifuged rats responded to erythropoietin. Cosmos flight animals' cells formed fewer CFU-E than the controls but this was reversed in the centrifuge studies. There were no essential differences in the erythropoietin levels of test groups as compared to control groups.

Blood volume and erythropoiesis in the rat during spaceflight.

A decreased red blood cell mass (RBCM) and plasma volume (PV) have been consistently found in humans after return from spaceflight. Rats flown on the Spacelab Life Sciences-1 mission were studied to assess changes in RBCM, PV, erythropoiesis, and iron economy. The RBCM and PV increased in both ground control and flight animals as expected for growing rats. However on landing day, both the RBCM and PV, when normalized for body mass, were significantly decreased in the spaceflight animals. During an 8-d postflight observation period, iron incorporation into circulating red blood cells was diminished in the flight animals. During the first 4 d postflight, increases in reticulocyte counts were significantly smaller in the flight than the control animals. Fewer erythropoietin-responsive progenitor cells were recovered from the bone marrow of flight animals after landing than control rats. Serum erythropoietin (EPO) levels were the same in both groups. Thus, rats subjected to a 9-d spaceflight had less increase in RBCM than controls and diminished erythropoiesis during an 8-d post-spaceflight observation period. The rat, like humans, appears to require a smaller blood volume in microgravity.

Antigenic heterology between flagellin and flagella of Bacillus subtilis.

The antigenic properties of Bacillus subtilis flagella and periodate-treated flagellin were compared by using immobilization-inhibition and diethylaminoethyl cellulose binding assay. The results suggest that there are both unique and cross-reactive antibodies elicited by B. subtilis flagella and flagellin.