A rat pulmonary alveolar macrophage (PAM) cell line (NR8383) was initiated in culture in the presence of a gerbil lung cell conditioned medium (GLCM), and has been propagated continuously for over 36 mo. When examined at different times throughout this in vitro period, NR8383 exhibited characteristics typical of macrophages: (a) Zymosan ingestion was seen in 90 to 98% of the cells examined; (b) Pseudomonas aeruginosa phagocytosis in 50 to 80%; (c) Nonspecific esterase activity in greater than 95%. During the first 6 mo., the PAM replicated with doubling times approximating 15 to 20 d. Throughout this period, GLCM dependence was evident. After 27 wk in vitro, NR8383 replication increased markedly, and within 2 wk, the doubling time was less than 48 h. NR8383 was readily monitored by [3H]thymidine (TdR) blastogenesis assay. In the presence of GLCM uptake of [3H]TdR was fivefold greater than in control cultures. Adherence and growth kinetics were effectively controlled by modulation of GLCM or serum content in culture medium. It was demonstrated that PAM growth factor(s) is ubiquitous, not species-specific, and under certain conditions may be derived from "endogenous" sources of persisting non-PAM populations within the parent, uncloned line NR8383. Cloned progeny remain devoid of non-PAM "feeder" cells, but retain macrophage properties, including interleukin-1 secretion, Fc receptors, and H2O2 production.
Summary 1-2% of adult mouse thymocytes express the T cell receptor ~/B (TCtL-oe/B) together with the interleukin (IL) 2P, g (p70), but not the o~ (p 55) chain. We show that the previously described ol/~/-TCR + CD4-8-and the partially overlapping Ly6C + thymocytes are contained within this subset. Most IL-2R~ + ol/B-TCR + cells have a mature and activated (heat stable antigen [HSA]-, thymic shared antigen 1 [TSA-1]-, CD44hig h, CD69 +) phenotype. Overrepresentation of V~8.2 in both CD4-8-and CD4 and/or CD8 + IL-2RB + thymocytes suggests that IL-2RB expression is induced by a TCR-mediated activation event. In mice transgenic for an H-2K bspecific TCR, IL-21~ + ceils were abundant under conditions of mainstream negative selection, i.e., in the presence of K b, but absent under conditions of mainstream positive selection or in a nonselecting environment. Together, these results show that in addition to clonal deletion, selfrecognition by immature thymoeytes leads to phenotypic maturation of a small subset of thymocytes expressing IL-2R~. IL-2-deficient mice contain normal numbers of IL-2R~ + oe/B-TCR + thymocytes, indicating that like mainstream T cell development, this minor pathway of positive selection does not depend on IL-2. However, in the absence of IL-2, the CD4/CD8 subset composition of IL-2RB + thymocytes is skewed towards CD4-8 +, mostly at the expense of CD4-8-. A possible relevance of this finding for the development of the immune pathology of IL-2-deficient mice is discussed.
Rabbit heterophil and human neutrophil primary granules contain sulfated glycosaminoglycans (GAGs) and acid phosphatase, which can be readily stained in immature but not mature lysosomes. To determine whether this loss of staining represents masking of reactive components or removal of these components, we examined rabbit heterophils to see if high-iron diamine (HID)-reactive sulfate and acid phosphatase staining reappears in phagocytic vacuoles. Rabbit heterophils, obtained by peritoneal hivage, were incubated in vitro with latex beads or Pseudomonas aeruginosa for 15-60 mm Pre-embedment HID staining was enhanced in thin sections of unosmicated specimens with thiocarbohydrazide and silver proteinate (TCH-SP). Phagocytosis oflatex beads or bacteria was progressively more prominent with time. Primary granules that were degranulated or in the process of degranulating into phagocytic vacuoles demonstrated intense sulfate staining (L). Heterophils incubated 60 minutes with-.. latex particles. N, nuclei. Bar = 1 pun.
Electron probe X-ray microanalysis (XRMA) of freeze-dried ultrathin sections provides the capability of measuring intracellular elemental content. This methodology was used to investigate the stimulus-permeability coupling responses associated with phagocytosis of Pseudomonas aeruginosa by cultured pulmonary alveolar macrophages (PAMs) of rats. PAMs were challenged with P. aeruginosa suspended in Gey's buffer at a bacteria to PAM ratio of 50:1 for 1 h at 37 degrees C. A 1-mm3 pellet of the unchallenged control PAMs, challenged PAMs and P. aeruginosa alone was quench-frozen in nitrogen-cooled, liquid propane, and 0.1-micron cryosections were cut at -100 degrees C. X-ray spectra were collected for nucleus and cytoplasm of 39 control PAMs, 36 challenged PAMs and 40 P. aeruginosa. Concentrations (mmole/kg dry weight) were obtained for Na, Cl, K, Ca, Mg, P, S for each cell. In the control PAMs, the content was similar to other mammalian cells. Moreover, there were no differences in elemental content between nucleus an cytoplasm. In the challenged PAMs, Na concentration was 4 times that of control PAMs (p less than 0.001) whereas Cl was double (p less than 0.001), K was 29% lower (p less than 0.001), and Ca was 4 times higher (p less than 0.05). The elemental concentration profile in the P. aeruginosa was distinctly different from that of the PAMs: higher Na, Ca, Mg, but lower Cl and K values. These results demonstrated elemental content changes in cultured PAMs challenged with P. aeruginosa that indicate a stimulus-permeability response by membranes associated with the phagocytic process.
Frozen sections of chicken lymphoid organs were examined for lymphocyte surface antigens by antisera to T and B lymphocytes (ATS;ABS), and for the presence of immunoglobulin (Ig) G Fc and complement receptors (FcR;CR) by hemadsorption with sheep erythrocytes (E) coated with chicken IgG(EA), and E coated with rabbit IgG and chicken complement (EAC). In the spleen FcR positive cells were confined to the periellipsoidal sheaths and the germinal centers. CR positive cells were found in the same spleen areas, as well as in the medulla of bursal follicles. These lymphoid areas reacted strongly with ABS, but they also stained with neutral alpha-naphthyl butyrate esterase, and phagocytosed carbon particles were found in the periellipsoidal sheaths. Furthermore, in vivo treatment with cyclophosphamide, which resulted in pronounced B-lymphocyte depletion, did not affect FcR activity, but reduced CR activity significantly. These data indicate that the FcR activity demonstrated in tissue sections is mainly confined to mononuclear phagocytes, while the CR positive cells are mainly B lymphocytes.
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