Flow cytometric measurement of telomere length.

The regulation of telomere length may be involved in the cellular physiology of senescence, reproduction, cancer, immune response to infection, and possibly immune deficiency. The measurement of telomere length, critical to research in this area, has traditionally been performed by Southern blot analysis, which is cumbersome and time consuming. Several alternative methods have been described in recent years. Some, such as pulsed-field electrophoresis, slot blots, and centromere-to-telomere ratio measurements are essentially improvements to the Southern blot technique. However, other methods such as fluorescent in situ hybridization on metaphase chromosome spreads and flow cytometry-based fluorescent in situ hybridization represent a completely new technical approach to the problem. In this review, we compare methods, with particular emphasis placed on flow cytometric techniques for measuring telomere length in situ and identifying potential areas where improvements may still be made.

M-CSF and GM-CSF promote alveolar macrophage differentiation into multinucleated giant cells with distinct phenotypes.

Multinucleated giant cells (MGC) are a hallmark of granulomatous reactions but the mechanisms that regulate their formation are unknown. To address this issue, we cultured resident alveolar macrophages (AM) from rat lung and examined the effects of defined cytokines on AM differentiation and MGC formation. The presence of MGC was found after 3 days in culture with maximal numbers obtained at 7 days and thereafter (up to 21 days). Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (25-75 U/mL) stimulated the formation of MGC (up to 4-fold), whereas interleukin (IL) -3, IL-10, and interferon-gamma (IFN-gamma) had no stimulatory effect. Interestingly, MGC with distinct phenotypes were observed in AM cultures: (1) spherical MGC with 3-16 nuclei, dense cytoplasm, and lower expression of beta3 integrin (Type 1) and (2) irregular MGC with 3-30 nuclei, thin and vacuolated cytoplasm, and higher expression of beta3 integrin (Type 2). Furthermore, the actions of M-CSF and GM-CSF on AM were found to be different. GM-CSF promoted, in AM cultures, the appearance of an elongated fibroblastoid phenotype and stimulated mostly the formation of Type 2 MGC. In contrast, M-CSF did not cause significant change in the general morphology of regular AM but stimulated the appearance of both Type 1 and Type 2 MGC. Reverse transcriptase-polymerase chain reaction analysis demonstrated that, under these conditions, M-CSF induced GM-CSF gene expression in AM. In addition, neutralizing antibodies against M-CSF selectively decreased the formation of Type 1 MGC, whereas neutralizing anti-GM-CSF inhibited Type 2 formation. These data suggest that M-CSF promotes AM differentiation into Type 1 MGC, whereas GM-CSF stimulates the formation of Type 2 and that M-CSF and GM-CSF may selectively regulate in an autocrine fashion AM differentiation into distinct MGC.

Alveolar macrophage inhibition of lung-associated NK activity: involvement of prostaglandins and transforming growth factor-beta 1.

Natural killer (NK) activity plays an important role in host defense. It is likely that this defensive role is shaped by compartmental and local environmental factors. The present study investigated the regulatory effects of alveolar macrophages (AM) on lung-associated NK activity. AM and lung lymphocytes (LL) were permitted to interact in a two-chamber system which prohibited cell contact but supported diffusion of soluble factors. AM were found to inhibit NK activity from LL in a time-dependent and reversible manner. The inhibitory event was shown to be mediated by soluble factors acting upon a post-binding event(s) in the lytic pathway of LL. AM inhibition was sensitive to indomethacin treatment (10(-5) M), which caused a decrease in prostaglandin E2 (PGE2) concentrations. Quantitation of PGE2 levels and treatment of LL with exogenous PGE2 indicated that the inhibitory effect could not be exclusively due to PGE2. It was subsequently found that exogenous transforming growth factor-beta 1 (TGF-beta 1) also inhibited LL NK activity and that treatment of inhibitory AM supernatant with a neutralizing antibody to TGF-beta 1 adsorbs up to 55% of its inhibitory activity. Moreover, the amount of TGF-beta 1 found in AM-LL co-culture media (25 pg/mL) correlated well with the level of NK inhibition observed. By contrast, platelet-derived growth factor and nitric oxide did not play a significant role in mediating AM suppression. Taken together, the data suggest that AM inhibit lung NK activity by interfering with post-binding lytic event(s) through the production of PGE2 and TGF-beta 1.

Effects of hyperthermia on natural killer cells: inhibition of lytic function and microtubule organization.

Cells with natural killer activity (NK) may play an important role in host defence against tumour cells. The lytic function of NK cells is very sensitive to hyperthermic inactivation. However, cells with NK activity isolated from rat spleen and exposed to 41-42.5 degrees C for 30 min could partially recover their cytotoxic activity after incubation at 37 degrees C. The recovered cytotoxicity was still NK-specific, as it only resulted in the lysis of YAC-1 sensitive targets, and could not lyse NK-resistant P815 mastocytoma cells. Conjugate formation assay using NK cells labelled with specific monoclonal antibody (mAb) 3.2.3 indicated that the binding of NK cells to targets was not significantly affected by heat treatment. Compared to controls, however, microtubule organizing centre (MTOC) reorientation towards the region of intercellular contact was reduced by 40% in heated effector cells. This was accompanied by a greater inhibition (62-77%) of NK lytic activity. Kinetic analysis indicated that MTOC reorientation capacity recovered following incubation at 37 degrees C. MTOC recovery was maximal 4 h after treatment whereas that of lytic activity peaked at 6 h. These data indicate that NK cells recover NK-specific lytic activity after heat inactivation. Moreover, our study demonstrates that hyperthermia interferes with post-binding MTOC reorientation, and further supports a role for microtubule in secretory processes involved in NK-mediated cytolysis.

Effects of biological response modifiers on lung natural killer activity.

Natural killer (NK) activity plays an important role in host defense against tumors, especially once augmented by immunomodulators. It is likely that the modulation of NK cells is a reflection of the environment in which they reside. The current study was undertaken to characterize the response profile of lung interstitial lymphocyte natural killer (LLNK) activity to various biological response modifiers (BRM) in vitro after short term incubation (18h). The presented data show that treatment of lung lymphocytes with human recombinant interleukin 2 (rIL-2), purified rat interferon alpha/beta (IFN-alpha/beta), or murine recombinant tumor necrosis factor alpha (rTNF-alpha) resulted in a dose-dependent increase in LLNK activity. The maximum stimulation was similar for rIL-2 and IFN-alpha/beta, although a much higher concentration of IFN-alpha/beta was required to reach this level of stimulation. The maximum response to rTNF-alpha treatment was about half that seen with rIL-2 or IFN-alpha/beta and it, too, required a high concentration. By contrast, rat recombinant interferon gamma (rIFN-gamma) or murine recombinant interleukin 1 (rIL-1) failed to alter LLNK activity significantly when used alone. Furthermore, doses of IFN-alpha/beta and rTNF-alpha that had little enhancing effect were able to synergize with a suboptimal dose of rIL-2, whereas rIL-1 and rIFN-gamma failed to do so. These data demonstrate the response of lung NK activity to BRM treatment, which is important for the responsible and effective use of BRM. However the spectrum of lung NK cell response to BRM is smaller than that previously reported for NK cells from other anatomic compartments.

Comparative analysis of natural killer function in lung, spleen, and peripheral blood lymphocytes: evidence of differential characteristics.

Organ associated as well as systemic natural killer (NK) activity play an important role in host defense. It is likely that tissue NK cells exhibit some specific characteristics resulting from their local environment. The purpose of the present study was to compare NK cell function of rat lung to that of spleen and blood in terms of magnitude of NK activity, NK cell number, and response to biological response modifiers (BRM). Lung interstitial lymphocytes (LL) were found to display significant cytotoxicity which was selective for YAC-1 tumor cells and abolished by treatment with antiasialo GM-1 serum and L-leucine methyl ester. In addition, LL were more effective than peripheral blood lymphocytes (PBL) and spleen lymphocytes (SL), although LL contained the same proportion of NK 3.2.3 positive cells than the spleen. Furthermore, the in vitro response to BRM varied both with BRM used and effector cell origin. Human recombinant interleukin 2 (rIL-2) increased NK activity of LL and PBL to a similar extent, whereas SL were more responsive to such treatment. Interferon alpha/beta (IFN-alpha/beta) increased NK activity in all three compartments with PBL being less responsive. By contrast, rat recombinant interferon gamma (rIFN-gamma) failed to augment LL or SL NK activity but did result in a significant enhancement of PBL activity. Together these data indicate that the lung contains comparable numbers of NK cells to that of spleen but which display higher specific activity. Furthermore, NK cell response to BRM vary with tissue origin, suggesting that tissue specific response may be an important determinant for the effective use of BRM.