Functional capacity of neutrophil granulocytes in deep-sea divers.

Neutrophil granulocytes (PMN) are main defenders against invading microbes. We evaluated the adaptive response of PMN from divers exposed for weeks to high total and oxygen pressures. Under these conditions PMN could be primed to give a heightened respiratory burst upon stimulation with the bacterial peptide analogue, formyl-methionyl-leucyl-phenylalanine (FMLP): blood PMN sampled both shortly after operational saturation dives offshore and during an onshore test-dive gave larger responses than control pre- or post-dive PMN from the same subjects and PMN from laboratory personnel. The assays used measured oxygen consumption, intracellular H2O2 availability, and chemiluminescence. The submaximal responses provoked by the non-metabolizable diacylglycerol analogue phorbol myristate acetate (PMA) were less and less often increased. Such enhanced PMN responsiveness may possibly decrease resistance to skin and other infections that are encountered in divers, if PMN thereby failed to localize correctly to inflamed tissues.

Thymus and T cells are not essential for rat leucopoiesis.

It is generally held that T lymphocytes take part in the regulation of haemopoiesis. We have examined whether, in vivo, the influence of thymus of functional T cells is dispensable for the steady-state or accelerated formation of granulocytes (and in some experiments macrophages), utilizing a rat model. Untreated normal and athymic 'nude' rats had similar blood and marrow granulocyte counts and marrow proliferative activity. Bone marrow regeneration after two cytotoxic insults to the two kinds of rats gave no clues to an important regulatory role for thymic factors or T cells. Nor were such clues obtained in experiments where bone marrow cells from normal rats were cultured in vivo in diffusion chambers (DC), in either normal rats or rats undergoing a graft-versus-host (GvH) reaction (with supranormal serum levels of cytokines). On the other hand, when marrow cells from athymic rats were similarly cultured in DC, small but significant differences in leucopoiesis occurred between the three kinds of DC hosts: Marrow cells lacking functional T cells generated fewer proliferative granulocytes and had a lower proliferative activity when cultured in athymic than in normal hosts, whereas the proliferative granulocytes were most numerous in chambers carried by GvH rats. No differences were found for macrophages and eosinophilic granulocytes. The results indicate that thymic hormones or T cells or both can stimulate granulopoiesis. They apparently play no indispensable role in short-term leucopoiesis, however, since their influence was weak in our experimental models. Consequently, marked effects seen in vitro in simplified cellular systems may lose some importance in the more complex in vivo setting.

A new method for rapid technetium-99m labelling of leucocytes: functional cell studies in vitro.

The aim of this study was to assess the cytotoxicity of a new leucocyte-labelling method, which may be used clinically to localize inflammatory and immune reactions. Human blood leucocytes, their mononuclear sub-population, and mouse mononuclear bone marrow cells were labelled with 99mTc for 30-45 min, washed once, and then evaluated in various functional assays. The new procedure includes [99mTc]-labelling with a bisalt method, in the presence of dihydroxybenzoic acid as an intermediate antioxidant-complexing stabilizer, and a carboxylic acid salt of stannous ions as a reducing agent. To challenge the method, cells were labelled about two orders of magnitude more heavily in these initial methodological studies than in on-going clinical trials. Labelled leucocytes ingested latex beads as readily as the controls, but migrated chemotactically and randomly somewhat slower than the control cells. The lymphocytes were triggered by PHA and Con A in a normal way. However, lymphocytes and haemopoietic progenitor cells exposed to radiation for several days, were killed by the isotope doses used, of which about 2% (i.e. 20 MBq) were bound per million cells. All deleterious effects were apparently due to irradiation, and the labelling procedure itself did not damage the cells.

Production of proliferation inhibitors by mature granulocytes.

The aim of the experiments was to find ways of increasing the yield of small molecular weight inhibitors of cell proliferation released by granulocytes. Almost pure populations of granulocytes from pig or human blood, or from sterile inflammatory exudates in rats were treated in various ways and then spun down. Molecules below approximately 10 000 dalton (Diaflo ultrafiltration or Sephadex G 25 filtration) in the supernatants were tested for inhibitory activity by measuring 3H-thymidine incorporation in 5 to 6-h coverslip cultures of rat bone marrow cells. The different granulocyte treatments were: Freeze-thawing, sonication, incubation (at +4 degrees -37 degrees C) in hypotonic media (0-200 mosm/kg), storage in vitro overnight (at +4 degrees C) before incubation, incubation at 37 degrees C in complete and buffered tissue culture medium (Fischer's with 10 mmol/1 HEPES), incubation in saline only (2-h periods, approximately 70 X 10(6) cells/ml), or with lidocaine added, with Ca++ and the Ca++ ionophore A-23187, with K+ and the K+ ionophore Valinomycin, with a high K+ concentration (50 mmol/1), with arachidonic acid, with a cAMP analogue, or with a protease inhibitor added during or at the end of the incubation. On a per cell basis rat peritonitis granulocytes released more inhibitor than pig blood granulocytes, whereas human blood granulocytes were not detectably inhibitory at all. Arachidonic acid was the most promising agent tested to increase inhibitor release above that occurring spontaneously from granulocytes incubated in saline.

Methodological studies on growth of mouse granulocyte/macrophage colonies in methylcellulose-containing media.

We studied the influence of various physicochemical parameters on colony development and total cell numbers in 7-day methylcellulose cultures of mouse bone marrow cells. Colony growth was markedly retarded by an increase of PO2 from approximately 6.7 kPa towards that in ambient air and by a decrease of incubator temperature from 37 degrees C to 33 degrees C. Medium osmolality above approximately 340 mosm/kg inhibited formation of granulocytes (in cultures containing growth factors from pokeweed mitogen-stimulated spleen cells), but not macrophages (L cell-produced growth factors). At most, colony growth was affected slightly by moderate changes in pH (7.17-7.47) or concentration of methylcellulose (0.77-1.02%), or by the presence of 2-mercaptoethanol (50 mumol/1), Hepes buffer (25 mmol/1), or erythropoietin (0.1-1 units/ml). The culture trays could be stored for one day at 4 degrees C in the incubation boxes before colonies or cells were counted.

Formation of granulocytes and macrophages in mouse bone marrow cultures exposed to various anaesthetics.

The effects of anaesthetics on mouse bone marrow colony growth in vitro were examined. The culture dishes were kept in boxes of stainless steel, so that the composition of the gas phase could easily be controlled. After 1 week of culturing, cell colonies were counted. The cells (macrophages and in one type of culture also granulocytes) were then washed out of the dishes and counted. Enflurane, as well as halothane, present in the gas phase at concentrations used clinically, decreased the number of colonies and cells in a dose-dependent fashion. However, intravenously administered drugs such as diazepam, fentanyl, alfentanyl, sufentanyl, thiopental and pentobarbital were not inhibitory at concentrations used in anaesthetic practice, but at least some of them depressed cell formation when high concentrations were used.