Dale F. Gruber

Bone Marrow Myelopoiesis in Rats After 10%, 20%, or 30% Thermal Injury

The high incidence of serious opportunistic infections that follow thermal injuries is well documented. Normal levels of functioning leukocytes are essential to the hosts ability to resist infection. This study examined alterations in murine granulopoiesis after the inducement of a standardized, sublethal, third-degree burn covering 10%, 20%, or 30% of the dorsal body surface area. Significant alterations arose in peripheral leukocyte concentrations after inducement of uncomplicated thermal injury. In general, within the first day of injury, all three trauma levels produced a peripheral leukocytosis that lasted for 35 days or more. The leukocytoses that followed 20% and 30% injuries were similar and in numerous respects paralleled previously reported human peripheral responses after similar levels of thermal trauma. Differential examinations of peripheral blood demonstrated the peripheral leukocytosis to be due primarily to the influx of morphologically mature-appearing polymorphonuclear neutrophils. Premature bone marrow release did not appear to be a factor as immature polymorphonuclear neutrophils were seldom greater than 2% of polymorphonuclear neutrophil totals. Bone marrow granulopoietic activity was examined by in vitro clonal cell culture techniques and assessed over a period of 35 days after injury. Granulocyte-macrophage colony forming cells (GM-CFC), indicative of marrow progenitor cell concentrations, were significantly increased for 28 to 35 days after 10% injury and 11 to 14 days after 20% or 30% injury. Normal or increased progenitor cell concentrations and a lack of morphologically appearing premature forms suggest that the leukocytosis is the result of injury-induced alteration(s) in polymorphonuclear neutrophil margination or release mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro modulation of canine polymorphonuclear leukocyte function by granulocyte-macrophage colony stimulating factor

Granulocyte-macrophage colony stimulating factor (GMCSF) promotes the growth of granulocytes and macrophages from undifferentiated bone marrow cells and modulates the oxidative responses of polymorphonuclear leukocytes (PMN) to endogenous chemoattractants. We found that,in vitro, naturally occurring glycolsylated human GMCSF does not disturb the resting canine PMN membrane potential, may attentuate PMN oxidative responses to PMA, and is, to a small degree, chemotaxigenic. GMCSF, however, inhibits PMN chemotaxis to zymosanactivated plasma (ZAP). Compared to temperature controls, GMCSF (1-100 U/ml) produced up to 1.5-fold increases in H2O2 production after 15 minutes, while phorbol myristate acetate (PMA) treated cells increased H2O2 production 8–12-fold after 15 minutes. Preincubation of cells with GMCSF (1–100 U/ml) prior to PMA stimulation significantly reduced the H2O2 levels induced by PMA. H202 production was inhibited up to 15% after 15 minutes of GMCSF preincubation and up to 40% after 60 minutes of preincubation. As a chemotaxigenic agent, GMCSF (10–1000 U/ml) was able to elicit 49%–102% increases in quantitative cellular migration, compared to random migration. Total cellular chemotaxis to GMCSF was < 30% of the response to ZAP. Preincubation of PMNs with GMCSF for 15 minutes significantly inhibited ZAP-induced cellular migration. Human GMCSF does not appear to activate canine PMNin vitro and may actually down-regulate PMN inflammatory responses.

Biochemical and Physiological Alterations in Canine Neutrophils Separated by Lysis or Percoll Gradient Isolation Technologies

Neutrophils were isolated from the peripheral blood of clinically normal canines by hypotonic lysis or density gradient (Percoll) centrifugation techniques. As a function of preparative technique, separated neutrophils were examined in vitro for alterations in membrane lipid integrity, and both granular and cytosolic specific enzymes. Membrane lipid disorder was assessed by merocyanine 540 (MC540), a fluorescent bioprobe, which intercalates into disrupted cellular lipid membranes. Evidence of membrane lipid disorder was based upon comparisons of mean cellular fluorescence exhibited by unstimulated cells. Based upon comparisons of mean cellular fluorescence the isolation methodologies did not appear significantly different. Significant levels of membrane disruption, due to preparative technique, were evident upon neutrophil stimulation with phorbol myristate acetate (PMA). Neutrophils which were Percoll-separated and PMA-stimulated demonstrated significant levels of membrane disruption not apparent in lysis-separated stimulated aliquots of cells. Cellular isolation methods did not significantly alter cellular myeloperoxidase (MPO) levels based on comparisons of cellular totals. Extracellular lactate dehydrogenase (LDH) levels of lysis-separated cells were four-times those of Percoll-separated neutrophils.

Modulations in Mouse Hemopoiesis after Engraftment with Lewis Lung (3LL) Carcinoma Cells

Hemopoietic changes in male C57BL/6Cum BR mice engrafted with Lewis lung carcinoma (3LL) were evaluated between day 7, when palpable tumors were present, to day 30 postengraftment. All experimental animals demonstrated decreasing hematocrits (down 40% by day 30) with concurrent leukocytosis which by day 30 postengraftment had reached levels 13.4 times normal. The myelocytic/erythrocytic ratio for normal animals was 1:3 (bone marrow: spleen). The ratio for engrafted animals ranged between 10:1 and 40:1. This apparent shift in production priorities is even more significant in light of the fact that femoral bone marrow cellularity had decreased by 33% on day 17. Splenomegaly, evident by day 7, was seven times control by day 17. Clonogenic analysis of erythroprogenitor cell concentrations revealed an inverse relationship between bone marrow and spleen. 27 days after engraftment, splenic populations demonstrated significant increases in colony forming unit-erythroid (115-fold), burst forming unit-erythroid (7.4-fold), whereas bone marrow concentrations had decreased (6-fold). This report suggests that initiation of 3LL tumor in mice results in a change in the degree of hematopoietic priorities and participation of erythroid organs.

Changes in canine neutrophil function(s) following cellular isolation by Percoll gradient centrifugation or isotonic lysis.

Analysis of cellular effector function(s) often requires their isolation from other cellular types. Cell separatory techniques could mask, or select out, clinically important functional lesions. We examined differences in canine peripheral blood neutrophil functions, i.e. migration and H202 production, following two commonly used cell separation techniques: isotonic lysis or density gradient (Percoll) centrifugation. Separation methodology was observed to have a significant impact on both metabolic and mobility functions. In comparison to isotonic lysis, Percoll separation caused near 100% increases in random migration, near 40% decreases in chemotaxis and 70% increases in H202 production.

Lymphomyelopoietic activity of submandibular gland-conditioned medium☆

Abstract Submandibular gland-conditioned medium (SMG-CM) possesses a number of lymphomyelopoietic regulatory features when examined for colony-forming units-culture (CFU-C), macrophage colony-forming cell(s) (M-CFC), and lymphoid mitogenic responses. Equal quantities of pregnant mouse uterine extract (PMUE) and SMG-CM demonstrated similar quantitative colony-stimulating abilities when examined on day 10 for CFU-C. The morphological appearance of those 10-day colonies was, however, distinctly different. The SMG-CM-stimulated cultures demonstrated only diffuse colony growth whereas PMUE-stimulated cultures showed conversely a 6:1 ratio of compact to diffuse type colonies. When examined on day 22 for M-CFC, SMG-CM-stimulated cultures decreased in number from the day-10 counts, and PMUE-stimulated cultures demonstrated a two-fold increase in number. Bone marrow cells in the presence of phytohemagglutinin (PHA) and SMG-CM increased the uptake of [3H]TdR 6-fold over control values. SMG-CM alone and CON-A plus SMG-CM increased [3H]TdR uptake by 30–40%. No difference was noted in lipopolysaccharide-(LPS) stimulated cultures with or without SMG-CM. Splenic responses to SMG-CM in the presence of PHA, CON-A, or LPS were all decreased from 20 to 75%.

Immunologic and hematologic perturbations in models of combined injury.

Many aspects of the reports in the literature describing anergic conditions that exist post trauma are confirmed by data gathered by the Armed Forces Radiobiology Research Institute in a comprehensive program to study combined-injury effects. Dealing with one aspect of this study, this article discusses the synergism of trauma and illness coincident with such stressors as whole-body irradiation, thermal injury, and sepsis. Experimentally produced sepsis depresses and delays both cellular and humoral response patterns in canine and murine modeling systems. Lyphoid populations, dependent on the time of collection post trauma and the organ source, may present an intially augmented response pattern, which within 48 hours becomes depressed to below-normal levels. In addition, immunologic anergy can be induced by the presence of bacterial products such as exotoxin-A. Further mechanisms and regimens of prophylactic immunomodulation are under investigation.

Submandibular gland-conditioned media suppression of mitogen induced T- and B-cells blastogenesis☆☆☆

Abstract Cells from a normal, non-hematopoietic organ population are demonstrated here for the first time to elaborate substances capable of suppressing lymphocyte blastogenesis. Pooled, cell-free conditioned media collected from cultures of male mouse submandibular glands (SMG-CM) suppress lymphoidal uptake of [ 3 H]TdR induced by mitogens — phytohemagglutinin (PHA), concanavalin-A (CON-A), and lipopolysaccharide (LPS). PHA- and CON-A-induced responses were suppressed by 34–40%. The LPS respondent population was suppressed by over 60%. The nature of the suppression was non-cytolytic and was time- and dose-dependent. Control populations of spleen cells receiving no mitogen treatment were stimulated by more than 50%.

Aminophylline induced oxidative metabolism in isolated canine polymorphonuclear leukocytes

Adenosine reportedly mediates myocardial and skeletal blood flow, bronchoconstriction, and cellular production of toxic oxygen radicals. Cellular effects of adenosine can be antagonized by the methylxanthines, which are widely used in the clinical treatment of obstructive airway diseases. Methylxanthine compounds such as aminophylline and theophylline inhibit the cyclic nucleotide phosphodiesterase of smooth muscle, reversing pathogenic states of bronchoconstriction. Recent techniques in flow cytometry allow examination of individual cells for the electrophysiological and metabolic cellular side effects of methylxanthine therapy. We report that the flow cytometric examination of isolated canine peripheral neutrophils, in the presence of therapeutic concentrations of aminophylline resulted in small but significant membrane depolarization and almost fivefold increases in baseline cytosolic H202 levels. If aminophylline is capable of direct in vitro activation of isolated canine neutrophils it may have the capacity to potentiate neutrophil activation in vivo: indirectly by competing with circulating modifiers, such as adenosine, for cell surface receptor sites and directly by the induction of toxic oxygen radicals as demonstrated here. H202 induction by aminophylline and other xanthine derivatives may become clinically important in instances of vascular occlusion, stasis, or instances of reperfusion where neutrophils may become activated. In an activated state, neutrophils could contribute to pathogenicity and tissue damage by indiscriminantly releasing oxygen-reactive species.