Blood Mononuclear Cell Cultures Research Articles

Human mononuclear cell factors mediate cartilage matrix degradation through chondrocyte activation.

Human blood mononuclear cells (BMC) in short-term culture secrete one or more factors that induce degradation of matrix proteoglycan and collagen in cartilage explants in organ culture. Induction of matrix degradation took place both in nasal septum and articular cartilage explants in the presence of the mononuclear cell supernates. Cartilage degradation in this system was absolutely dependent on the presence of live chondrocytes. Matrix depletion did not occur in dead cartilage explants cultured with active supernates. Supernates obtained from unstimulated BMC showed variable cartilage matrix degrading activity (MDA). BMC stimulated with phytohemagglutinin (PHA) showed increased MDA, which in one dilution experiment was found to be five times higher than that in the unstimulated control supernate. Concanavalin A and pokeweed mitogen were also shown to stimulate release of MDA. Time experiments showed that most of the degrading activity was released by the mononuclear cells during the first day of culture. The cellular origin of MDA was investigated with the aid of partially purified BMC subpopulations. Removal of adherent cells resulted in a decrease of MDA release. Purified T lymphocytes failed to show enhanced MDA release in spite of their ability to mount a virtually intact proliferative response to PHA. Purified adherent cells also failed to show enhanced PHA-dependent MDA release. Nevertheless, restoration of PHA-dependent MDA release took place in reconstituted cell populations containing both T lymphocytes and monocytes. These experiments suggest that MDA may be released by adherent mononuclear cells, presumably monocytes, and that the PHA-dependent increase in MDA release may be mediated by T lymphocytes. Partial characterization of MDA by gel chromatography showed one active fraction corresponding to an apparent molecular weight ranging from 12,000 to 20,000. The fraction was also shown to degrade cartilage matrix only in the presence of live chondrocytes. These results demonstrate that factors released by human BMC mediate degradation of matrix proteoglycan and collagen in intact cartilage explants through chondrocyte activation. This pathogenic mechanism may play a role in in vivo cartilage destruction in chronic inflammatory joint diseases.

Myeloid Differentiation of Human Blood Mononuclear Cells in Liquid Culture

Human blood mononuclear cells were prepared from peripheral blood or single donor plateletpheresis residues by depletion of adherent cells and T lymphocytes. In double-layer soft agar culture, 5 x 10(5) such cells yielded from 33 to 165 colonies. For liquid culture, these cells were suspended in McCoy's 5A medium with 15% fetal bovine serum and 0%, 20%, or 40% conditioned medium (CM) and incubated for up to 14 days at 37 degrees C in a humidified 5% CO2-95% air atmosphere. The number of cells in cultures with CM decreased about 0%-10%, while cell counts from cultures without CM decreased about 45%-65%. In cultures with CM, 5%-20% of the cells were classified as blasts after 3-5 days. After 7-11 days, blasts and promyelocytes comprised up to 53% of all cells. After 9-11 days, cells with specific granules and maturing nuclei comprised up to 56% of all cells. At 11 days, up to 66% of the cells contained peroxidase-positive granules. Cultures without CM contained no more than 5% blasts and promyelocytes and less than 5% maturing granulocytic cells. 3H-thymidine and Na235SO4 incorporation reached a peak at 3-5 days and at 5-11 days, respectively, in cells from cultures containing CM.

Myeloid differentiation of human blood mononuclear cells in liquid culture

Human blood mononuclear cells were prepared from peripheral blood or single donor plateletpheresis residues by depletion of adherent cells and T lymphocytes. In double-layer soft agar culture, 5 x 10(5) such cells yielded from 33 to 165 colonies. For liquid culture, these cells were suspended in McCoy's 5A medium with 15% fetal bovine serum and 0%, 20%, or 40% conditioned medium (CM) and incubated for up to 14 days at 37 degrees C in a humidified 5% CO2–95% air atmosphere. The number of cells in cultures with CM decreased about 0%-10%, while cell counts from cultures without CM decreased about 45%-65%. In cultures with CM, 5%-20% of the cells were classified as blasts after 3–5 days. After 7- 11 days, blasts and promyelocytes comprised up to 53% of all cells. After 9–11 days, cells with specific granules and maturing nuclei comprised up to 56% of all cells. At 11 days, up to 66% of the cells contained peroxidase-positive granules. Cultures without CM contained no more than 5% blasts and promyelocytes and less than 5% maturing granulocytic cells. 3H-thymidine and Na235SO4 incorporation reached a peak at 3–5 days and at 5–11 days, respectively, in cells from cultures containing CM.

Identification of collagenase in cultured blood mononuclear cells.

Mononuclear cells isolated from peripheral whole blood were fractionated by erythrocyte-rosetting into T cells and non-T cell subpopulations which were tested for collagenase-producing capacity. Mononuclear cells and purified T lymphocytes, as well as non-T (B) lymphocytes, produced collagenase during serum-free culture. Removal of the macrophages from the non-T cells did not affect collagenase production by lymphocytes.

Adult and fetal hemoglobin production in erythroid colonies from subjects with beta-thalassemia or with hereditary persistance of fetal hemoglobin (HPFH).

The synthesis of alpha and non-alpha chains (beta, delta, G gamma, and A gamma) was studied in cultures of peripheral blood mononuclear cells from eleven beta-thalassemia heterozygotes, two HPFH heterozygotes, and one HPFH homozygote. The synthesis of Hb F in the thalassemia colonies (average value: 12.6%) was comparable to that in normal adult colonies (average value: 12.6%) was comparable to that in normal adult colonies (average value: 12.2%). The percent G gamma chain in the Hb F varied greatly but a relationship between the G gamma chain percentage in the Hb F from colonies and that from peripheral blood was established. The relative synthesis of Hb A2 in colonies of beta-thalassemia heterozygotes (average value: 5.8%) was 1.6 times as much as that in colonies of normal adults (average value: 3.6%). Hb A2 and Hb A were absent in the colonies of the HPFH homozygote. The alpha/non-alpha (i.e., beta, gamma, and delta) ratio of the hemoglobins in the cultured cells of the beta-thalassemia heterozygotes and the alpha/beta and alpha/beta ratios of isolated Hb A and Hb A2 were about one (range 0.74 to 1.38). The alpha/gamma ratio of the Hb F synthesized in BFUe-derived colonies of the HPFH homozygote, however, was 1.5. These results suggest a deficiency in the in vitro culture system resulting in decreased levels of alpha-mRNA or in a partial inhibition of initiation of protein synthesis which is known to reduce the synthesis of alpha chains more than that of the beta chains.

Characteristics of suppressor cell activity appearing in cocultures of two individuals with immunodeficiency with thymoma.

Suppressor cell activity in two individuals (S1 and S2) with immunodeficiency with thymoma (ID-THY) was studied in peripheral blood mononuclear cells in pokeweed mitogen-stimulated single culture and cocultures. Secreted Ig was measured by radioincorporation and immunoprecipitation after 5-7 days. Control cocultures of normal/normal cells showed, in most cases, a percentage observed to expected ratio (% O/E) of cpm Ig near 100%. However, augmentation (% of O/E > 150) was often encountered, whereas suppression (% O/E < 50) was found only once in forty-two cocultures. In ID-THY/normal cell cocultures the degree of suppression by ID-THY cells varied widely when the same or different cocultivants were retested. This finding could be explained in part by an inverse correlation between the amount of secreted Ig produced by normal cells in single culture and the degree of suppression of the same normal by ID-THY cells in cocultures. A panel of normal cells were all suppressed when a range of S1 or S2/normal cell ratios were tested, weighing against genetic differences in suppressibility in the above system. ID-THY cells failed to block Ig secretion of human lymphoblastoid line cells, suggesting that the mechanism of suppression was related to a block in differentiation rather than interference with Ig synthesis per se. An experiment using cocultivants separated by a Millipore membrane showed that suppression was mediated by a humoral factor.

Rheumatology: An Annotated Bibliography of Recent Literature

Rheumatology: An Annotated Bibliography of Recent Literature

Prostaglandin production by rheumatoid synovial cells: stimulation by a factor from human mononuclear Cells.

Human peripheral blood mononuclear cells (lymphocyte-monocyte) in culture release a solube factor which can stimulate, up to 200-fold, production of prostaglandin E2 by isolated, adherent, rheumatoid synovial cells. Production of the factor by the mononuclear cells is enhanced by phytohemagglutinin. This factor is similar in apparent mol vt (10,000-20,000) to that which also stimulates collagenase production by the same cells.