Cytokine production by cryopreserved human peripheral blood mononuclear cells in response to periodontal pathogens.

Freezing techniques provide a way of repeating and extending immunological assays by using frozen portions of an individual's peripheral blood mononuclear cell fraction. Earlier work shows that the lymphocytes that are stored frozen retain their ability to respond to polyclonal B-cell activators, mitogens, superantigens and bacterial extracts of oral interest. These studies extend previous findings by determining cytokine production by lymphocytes following frozen storage for up to 24 weeks. Production of interleukin (IL)-1beta, IL-2, IL-6, and tumour necrosis factor (TNF)-beta by stimulated lymphocytes after cyropreservation was not significantly different from those responses before storage, with one exception: IL-6 production was negligible after 24 weeks' frozen storage when thawed cells were cocultured with pokeweed mitogen. After stimulation with extracts from Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, the proliferative capacity of the frozen cells was maintained as well as the production of IL-1beta, IL-2, and IL-6. TNF-beta was not produced in response to bacterial antigen stimulation. The ability of peripheral blood mononuclear cells to retain functional activity after frozen storage should permit more effective monitoring during longitudinal clinical studies and a better evaluation of changes in cytokine production in patients with advanced periodontitis both during and after treatment.

Interleukin-6 in neutrophils from peripheral blood and inflammatory periradicular tissues.

Periradicular tissue samples were recovered from patients undergoing endodontic surgery. Immunohistochemical procedures were used to identify interleukin-6 (IL-6) present in the neutrophils associated with these lesions. Using specific polyclonal antibody, 15 to 20% of the neutrophils associated with the periradicular tissue lesions were positive for IL-6. IL-6-positive plasma cells and histiocytes were also abundant. Cytokine production by neutrophils from the peripheral blood of nondiseased individuals was also evaluated. The cells were stimulated with lipopolysaccharides and bacterial extracts of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Significant levels of IL-6 were produced by cells stimulated with Escherichia coli lipopolysaccharide B5 (933 pg/ml/10(7) neutrophils) and E. coli lipopolysaccharide B12 (791 pg/ml/10(7) neutrophils) when compared with unstimulated cells (39.31 pg/ml/10(7) cells). There was a dose-dependent increase in IL-6 production when cells were stimulated for 24 h with 6 and 12 micrograms/ml of A. actinomycetemcomitans extract. Less stimulation occurred with P. gingivalis, but it was also dose-dependent. Neutrophils resident in periradicular lesions may, therefore, provide a source of IL-6 in addition to that produced by tissue histiocytes and plasma cells.

Studies of proliferative responses by long-term-cryopreserved peripheral blood mononuclear cells to bacterial components associated with periodontitis.

Freezing techniques provide a means for repeating and extending immunological assays with frozen aliquots of an individual's peripheral blood mononuclear cell fraction. Lymphocytes which are stored frozen for a limited time retain their ability to respond to polyclonal B-cell activators, mitogens, and antigens of dental interest. Our studies extend these previous findings by determining lymphocyte functional activity following frozen storage for up to 100 weeks. In addition, the autologous immune response was measured by spontaneous lymphocyte proliferation following 0, 1, 40, and 60 weeks of frozen storage. Peak responses for all individuals occurred at day 7 of incubation. The lymphocyte proliferative response to the superantigens toxic shock syndrome toxin-1 (TSST-1) and Staphylococcus enterotoxin A (SEA) were not changed after 100 weeks of frozen storage. Maximum responses varied among the individuals but occurred at equivalent stimulator concentrations. However, slopes generated from data obtained following 0, 4, 13, 20, 30, 50, 88, and 100 weeks of frozen storage showed no significant deviation from zero (P > 0.05) for all individuals tested. After 100 weeks of storage, the total changes in proliferative activity (counts per minute per week) were -2.1% +/- 16.8% and -5.5% +/- 17.0% for TSST-1 and SEA, respectively. The lymphocyte proliferative responses to pokeweed mitogen, concanavalin A, and sonicates of two periodontal pathogens (Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans) following frozen storage were similar to those with TSST-1 and SEA. These results indicate that peripheral blood mononuclear cells stored frozen may serve as appropriate controls to monitor changes in the disease state long-term periodontal treatment.

Changes in serum triiodothyronine kinetics and hepatic type I 5'-deiodinase activity of cold-exposed swine.

Swine exposed to cold air have elevated serum values of total triiodothyronine (TT3) and free T3 (FT3). To characterize the mechanism of these increases, we measured in vivo kinetic parameters after a bolus intravenous injection of 125I-labeled T3 by use of both multicompartmental (MC) and noncompartmental (NC) methods and in vitro hepatic type I iodothyronine 5'-deiodinase (5'D-I) activity. Ten ad libitum-fed 5-mo-old boars were divided into two groups, living for 25 days in either control (22 degrees C) or cold (4 degrees C) conditions. Cold-exposed animals consumed 50% more calories than control animals but showed no difference in total body weight, percent body fat, or plasma volume. Thyroid gland weight was increased 86% (P < 0.004), as was serum total thyroxine (TT4) (48%), free T4 (FT4) (61%), TT3 (103%), and FT3 (107%), whereas serum thyrotropin (TSH) was not different in cold-exposed compared with control animals. The T3 plasma clearance rate was similar between groups when both MC and NC techniques were used. However, T3 plasma appearance rate (PAR) was elevated in cold-treated animals 110% over controls by MC (P < 0.001) and 83% by NC methods (P < 0.001). The animal total hormone pool of T3 was increased 76% (MC) and 53% (NC) compared with control (P < 0.01). The Michaelis constant of hepatic 5'D-I was not different between groups, but the maximum enzyme velocity increased (106%; P < 0.02). Therefore cold exposure for 25 days is associated with increased energy intake, thyroid size, T3 PAR, and hepatic 5'D-I activity with little change in serum TSH.

Changes in triiodothyronine (T3) mononuclear leukocyte receptor kinetics after T3 administration and multiple cold-air exposures.

Repeated cold-air exposures increase human triiodothyronine (T3) plasma clearance rates. To study the response of the nuclear T3 receptor (NT3R) in this condition, binding characteristics were analyzed in human mononuclear leukocytes (MNL). In addition, we supplemented one group of individuals with a daily oral replacement dose of T3 to isolate the influence of serum thyroxine (T4) and thyrotropin (TSH) levels on receptor kinetics. The subjects were exposed to cold air (4 degrees C) twice/d, 30 min/exposure, for a total of 80 exposures. The T3-subjects received placebo [n = 8] and the T3 + subjects received T3 (30 micrograms/d) [n = 8] in a double-blind fashion. Mononuclear leukocytes were isolated from peripheral blood before the cold exposure and drug regimen began, and then after every 20 exposures. The dissociation constant (Kd) and maximum binding capacity (MBC) of the NT3R values were log transformed to minimize between-subject variability. In the T3+ group, serum total thyroxine (TT4), free T4 (FT4), and TSH were approx 50% lower than both basal and T3-values. The log10Kd increased 0.304 +/- 0.139 (p < 0.04) and the log10MBC increased 0.49 +/- 0.10 (p < 0.001) in the T3+ subjects compared to baseline. This change in MBC represents a 311% increase in the MBC over baseline and a fivefold increase over placebo-treated subjects. The T3- group showed no change in MBC over the study. These results describe for the first time the rapid modulation of the NT3R in response to the combined influence of cold exposure and reduced circulating T4 and TSH.

Relationship between changes in serum thyrotropin and total and lipoprotein cholesterol with prolonged Antarctic residence.

Antarctic residence (AR) is associated with a 50% increase in the thyrotropin (TSH) response to TSH-releasing hormone (TRH) and an expanded triiodothyronine (T3) distribution volume and extravascular hormone pool, collectively called the polar T3 syndrome. To investigate the possible biologic significance of this syndrome, we studied the relationship between nonstimulated TSH and serum lipid profiles in nine subjects, once while in California and monthly during 9 months of AR. We measured serum levels of TSH, total thyroxine (TT4), free T4 (FT4), total T3 (TT3), free T3 (FT3), thyroid-binding globulin (TBG), total cholesterol (T-CHOL), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), dietary cholesterol (D-CHOL), dietary fat (D-FAT), and dietary kilocalories at each month. The paired mean monthly change from baseline was used to determine significance. The group's mean levels of TSH (approximately 30%), TBG (approximately 16%), T-CHOL (approximately 4%), HDL-C (approximately 10%), and D-CHOL (approximately 19%) increased with AR (P < .05). Small but significant decreases (P < .05) were observed in the mean changes of TT4 (approximately 8%), FT4 (approximately 6%), and TT3 (approximately 6%). FT3, D-FAT, dietary kilocalories, body weight, TG, and the calculated low-density lipoprotein (LDL-C) were unchanged with AR. A significant rate of change (P < .05) during AR was also calculated from the slope of a fitted logarithmic function for TSH (0.96 +/- 0.31 mU.L-1 x mo-1), TBG (61.19 +/- 12.29 nmol.L-1 x mo-1), TT3 (0.09 +/- 0.04 nmol.L-1 x mo-1), TT4/TBG (-0.06 +/- 0.01/mo), TT3/TBG (-8.49 +/- 1.98 x 10(-4)/mo), and TG (-0.33 +/- 0.15 mmol.L-1 x mo-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple cold air exposures change oral triiodothyronine kinetics in normal men.

The influence of cold exposure on triiodothyronine (T3) kinetics was studied in 16 men before, during (biweekly), and after 80 (10/wk) cold (4 degrees C) air exposures. We used serum values before and up to 24 h after a pharmacological oral (o) dose of T3 [76.8 nmol (50 micrograms)] to calculate noncompartmental kinetic parameters. To assess the role of thyroxine (T4) and thyrotropin (TSH), we administered a replacement dose of T3 [46.0 nmol/day (30 micrograms)] to eight men (+T3 group) and placebo to eight others (-T3 group) for the 2-mo study. There was no group effect of T3 treatment (+T3) on oral total volume of distribution (TVdo), metabolic clearance rate (MCRo), or disposal rate (DRo). TVdo was not changed over the study. Cold increased MCRo by 5.4 +/- 2.0 l.day-1.m-2. DRo increased with cold by 10.2 +/- 4.4 nmol.day-1.m-2. Although serum TSH, total T4, and free T4 decreased by approximately 50% in the +T3 group, the changes in MCRo and DRo with cold were not different from those in -T3. We describe that human T3 kinetics are changed with brief repeated exposures to cold air and that these increases in MCRo and DRo do not appear to be dependent on TSH or T4.

Human cold air habituation is independent of thyroxine and thyrotropin.

Thyroxine (T4) is required in species possessing brown adipose tissue (BAT) for the maintenance of cold tolerance and adaptation. In humans, who possess negligible quantities of BAT, the importance of T4 has not been demonstrated. We studied the effects of decreased serum T4 and thyrotropin (TSH) on human cold habituation after repeated cold air exposures. Eight men (T3+) received a single daily dose of triiodothyronine (T3; 30 micrograms/day), and another eight men (T3-) received a placebo. All 16 normal thyroid men underwent a standardized cold air test (SCAT) under basal conditions in January and again in March after eighty 30-min 4.4 degrees C air exposures (10/wk). Measurements of basal metabolic rate (BMR), O2 consumption (VO2), mean arterial pressure (MAP), plasma norepinephrine (NE), serum TSH, free and total T4, and free and total T3 were repeated before and after 8 wk of exposure. TSH, free T4, and total T4 were 50% lower for T3+ than for T3- subjects. Total and free T3 were not different between groups. BMR was unchanged after habituation, whereas the cold-stimulated VO2, MAP, and NE were significantly reduced for all subjects in March. The relationship between VO2 and NE (r2 = 0.44, P less than 0.001) during the initial SCAT was unchanged with habituation. We suggest that human cold habituation is independent of major changes in circulating T4 and TSH.

Hematological parameters are altered during cold air exposure.

Whole blood hematocrit (HCT) decreases during multiple exposures to cold air. To better understand this finding, we have analyzed hematological profiles in 27 normal adult men exposed repeatedly to cold air in one of two experimental protocols. Experiment I was a cold air acclimatization study (CAA) conducted with two groups of 8 men in each group before, during, and after 80 separate 30-minute cold (4 degrees C) air exposures. As part of a metabolic study, half of the men received placebo daily (n = 8), and the other half received an oral daily maintenance dose of the thyroid hormone triiodothyronine (T3) (30 micrograms/day). Blood was analyzed prior to and after every 20 cold exposures. Both groups reacted similarly. When compared with basal conditions, hematocrit (HCT) and erythrocyte counts (RBC) were decreased (p less than 0.05); mean corpuscular hemoglobin concentration (MCHC) and plasma volume (PV) were increased with cold exposure (p less than 0.05). Hemoglobin (Hb), leukocyte counts (WBC), and mean corpuscular volume (MCV) were unchanged. Experiment II was carried out with 9 military volunteers during extended arctic winter field operations (EAO) in Utah and Alaska. Blood was analyzed prior to and after completion of EAO. A changing hematological profile similar to that in the CAA protocol was found. Hematocrit and RBC were decreased (p less than 0.02); MCHC and PV were increased (p less than 0.02). Hemoglobin, WBC, and MCV were unchanged. In addition, there was a negative correlation between HCT and the absolute reticulocyte count in this second experiment. It would appear that in instances of cold stress, whether induced or naturally occurring, certain blood cellular elements respond in a similar adaptive manner.

Radioprotection by polysaccharides alone and in combination with aminothiols.

We demonstrated that glucan, a beta-1,3 polysaccharide immunomodulator, enhances survival of mice when administered before radiation exposure. Glucan's prophylactic survival-enhancing effects are mediated by several mechanisms including (1) increasing macrophage-mediated resistance to potentially lethal postirradiation opportunistic infections, (2) increasing the D(o) of hematopoietic progenitor cells, and (3) accelerating hematopoietic reconstitution. In addition, even when administered shortly after some otherwise lethal doses of radiation, glucan increases survival. Glucan's therapeutic survival-enhancing effects are also mediated through its ability to enhance macrophage function and to accelerate hematopoietic reconstitution; glucan's therapeutic potential, however, is ultimately dependent on the survival of a critical number of hematopoietic stem cells capable of responding to glucan's stimulatory effects. Preirradiation administration of the traditional aminothiol radioprotectants WR-2721 and WR-3689 has been previously demonstrated to be an extremely effective means to increase hematopoietic stem cell survival. Therapeutic glucan treatment administered in combination with preirradiation WR-2721 or WR-3689 treatment synergistically increases both hematopoietic reconstitution and survival. Such combined modality treatments offer new promise in treating acute radiation injury.

Propranolol fails to lower the increased blood pressure caused by cold air exposure.

Cold air exposure stimulates a rise in mean arterial blood pressure (MAP) and plasma norepinephrine (NE). The specific contribution of the beta-adrenergic receptor to this pressor response is unknown. Therefore, we pretreated 12 normal men with placebo or a bradycardia-inducing amount of propranolol prior to exposing them to either 25 degrees C or 4 degrees C air. At 25 degrees C, propranolol pretreatment lowered heart rate (HR) and MAP. When we compared changes in MAP after their respective 30-min exposure at 25 degrees C and 4 degrees C, the cold elevated MAP by 18.4 +/- 1.5 mm Hg in subjects pretreated with propranolol compared with 13.0 +/- 2.5 mm Hg in subjects pretreated with placebo. Fingertip skin temperature (Tfing) measured at 4 degrees C, 9.5 +/- 0.8 degrees C in propranolol-pretreated subjects was lower than the 11.1 +/- 0.7 degrees C with that of placebo. Plasma NE increased equally during cold exposure with both placebo and propranolol pretreatment. We conclude that the beta receptor plays a minor role in generating the pressor response to cold air. Therefore, the effectiveness of acute administrations of propranolol for maintaining normotension in subjects exposed to cold environments may be attenuated.

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 zymosan-activated 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. H2O2 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 less than 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 PMN in vitro and may actually down-regulate PMN inflammatory responses.

Decreased free fraction of thyroid hormones after prolonged Antarctic residence.

We investigated the effects of Antarctic residence (AR) on serum thyroid hormone and cardiovascular responses to a 60-min standard cold air (0 degree C) test (SCAT). Serum total thyroxine (TT4) and serum total triiodothyronine (TT3), free T4 (FT4) and T3 (FT3), thyrotropin (TSH), and percent free fraction of T4 (%FT4) and T3 (%FT3) were measured in normal men (n = 15) before and after each of three SCATs. The SCAT was first carried out in California and then repeated after 24 and 44 wk AR. Mean arterial pressure (MAP) and sublingual oral temperature (Tor) were measured before and during each SCAT. The SCAT did not alter thyroid hormones before or after AR. The %FT4 decreased from 0.0334 +/- 0.0017 to 0.0295 +/- 0.0007% (P less than 0.002) with 44 wk AR but without a significant change in TT4 or FT4 for the same period. The %FT3 also decreased from 0.2812 +/- 0.0128 to 0.2458 +/- 0.0067% (P less than 0.005) after 44 wk AR. FT3 decreased (P less than 0.003) but TT3 and TSH were unchanged with 44 wk AR. The decrease in %FT4 and %FT3 may be theoretically accounted for by a 10% increase in either the capacity or the affinity of the serum binding proteins. The SCAT in California increased MAP and did not change Tor. After 44 wk AR, the SCAT no longer increased MAP but did lower Tor. The shift in the Tor and MAP response to the SCAT is consistent with the associated occurrence of cold adaptation during AR. We describe for the first time a decrease in the free fraction of both serum T3 and T4 present with extended polar residence and independent of a SCAT, further characterizing the recently reported "polar T3 syndrome."

Quantitative and functional alterations of peripheral blood neutrophils after 10% and 30% thermal injury.

Polymorphonuclear leukocytes (PMNs) comprise the majority of early nonspecific inflammatory responses to infection or trauma and, as such, must be of sufficient number and qualitative function to properly limit and combat inflammation. Peripheral PMNs isolated from rats that received 10% or 30% body surface area full-thickness thermal injuries were quantitated and examined for functional alterations in membrane potential and cytosolic hydrogen peroxide production for 35 days after thermal injury. With 10% thermal injury, leukocytes increased quantitatively to experimental maximums that were 70% above normal on day 7 before a return to normal by day 28. Platelet levels showed a nonsignificant decrease for 2 days after thermal injury before increasing to levels 20% to 40% above normal through day 28. Phorbol myristate acetate-induced PMN membrane depolarization was inhibited as much as 30% for 21 days after 10% thermal injury. No changes in oxidative activity were apparent except for day 14, when hydrogen peroxide production was 40% above normal. With 30% thermal injury, leukocyte quantities were three to five times normal, with increased relative numbers of PMNs and decreased lymphocytes through day 28. Platelet levels decreased for 4 days before increasing to levels 30% to 47% above normal through day 21. Compared with 10% thermal injury, 30% thermal injury further reduced the ability of PMN membranes to depolarize through day 35. In addition, PMN hydrogen peroxide production was 30% lower on day 1 and increased thereafter to levels that were 40% above normal on day 21.

Hypermetabolic priming of canine neutrophils by 7-S nerve growth factor.

Canine circulating neutrophils, isolated by a blood lysing technique, were incubated with 7-S nerve growth factor (NGF), at final concentrations between 12.5 and 800 ng/ml, for 30 minutes at 37 C. Neutrophil cytosolic H2O2 production, measured by flow cytometry, after 7-S NGF incubation was not significantly different from that produced at 37 C (baseline temperature controls) alone. Phorbol myristate acetate (PMA; 100 ng/ml) stimulation of neutrophils produced cytosolic H2O2 concentrations almost 13 times that of baseline temperature control neutrophils. Preincubation of neutrophils with 7-S NGF (100 to 800 ng/ml, 30 minutes, 37 C) and subsequent stimulation by PMA resulted in augmented H2O2 production in excess of twice that of neutrophils treated with PMA alone, and almost 30 times that of baseline temperature controls.

Effects of collection methods and storage on the in vitro stability of canine plasma catecholamines.

Norepinephrine (NE) and epinephrine (EPI) collected from dogs were sequentially and temporally measured in blood and plasma at 24 C. Heparin and EDTA anticoagulants, in combination with reduced glutathione and EDTA as a preservative, were also compared. Norepinephrine and EPI concentrations were measured by high-pressure liquid chromatography with electrochemical detection. In heparinized plasma, NE and EPI concentrations were relatively stable in the absence or presence of preservative after 24 hours at 24 C. In EDTA plasma, NE and EPI values were less stable when compared with those in heparinized samples. Norepinephrine concentrations in EDTA plasma without preservative decreased by 163.2 +/- 8.88 pg over 24 hours, compared with an 86.6 +/- 7.92 pg loss of NE in heparinized plasma. The degradation of EPI in EDTA plasma without preservative was also twofold greater, compared with that in heparinized plasma. Addition of preservative had no stabilizing effect on NE or EPI in heparinized or EDTA plasma. During long-term storage at -70 C, plasma NE and EPI values decreased less than 0.6 and less than 0.1 pg/d, respectively. Norepinephrine and EPI values were stable in heparinized blood for 6 hours but decreased to less than 25% and less than 6% of initial base line values, respectively, when plasma separation was delayed 24 hours.

In vitro effects of leukotriene B4 (LTB4) on canine PMN effector function(s).

The canine has become an accepted research model for the examination of a number of human clinical conditions. Despite it's status as a research model, little is known regarding the peripheral effects of inflammatory mediator substances. Products of arachidonic acid metabolism (leukotrienes) are reported capable of altering leukocyte functions. Because of the emerging importance of the canine research model and leukotrienes we examined the effects of leukotriene B4 (LTB4) on several in vitro functions of isolated canine peripheral polymorphonuclear leukocytes (PMN). Changes in forward angle light scatter properties of the cells were used as one measure of PMN activation. Other functional changes examined following LTB4 pretreatment included chemotactic capability, the electrophysiological state of the cell plasma membrane, and the metabolic oxidative response (i.e. H2O2 production). Random cellular movement of PMNs increased by 120% and 72% following preincubation with 10(-7) and 10(-9) M LTB4, respectively. LTB4 between 10(-7) and 10(-13) M did not significantly alter cellular resting membrane potential. Between 10(-7) and 10(-9) M LTB4 elicited significant levels of cellular H2O2 production. Although significant, H2O2 production was less than 40% that induced by phorbol myristate acetate (PMA). In numerous respects, canine in vitro PMN responses parallel previous reports of human cell function(s) in the presence of inflammatory mediators and may represent an attractive alternative for investigation of PMN dysfunctions.

Alteration of rat polymorphonuclear leukocyte function after thermal injury.

One portion of host defense to bacterial challenge(s) involves the activation and infiltration of endogenous polymorphonuclear leukocytes. Thermal injuries are frequently associated with immunologic abnormalities including alterations of polymorphonuclear leukocyte-associated nonspecific resistance. We examined isolated peripheral rat polymorphonuclear leukocytes for alterations in membrane potential, oxidative capability, and locomotor function after the experimental application of 20% full-thickness body surface area thermal injury. Thermal injury resulted in significant reductions of peripheral red blood cell concentration(s) and increases in leukocyte and platelet concentrations for 42 days after injury. In addition to the quantitative changes, polymorphonuclear leukocytes also demonstrated altered qualitative functions. Compared with phorbol myristate acetate-induced activation of normal cells, polymorphonuclear leukocyte membranes from thermal-injured animals were electrophysiologically less responsive for 3 weeks after injury. The ability of polymorphonuclear leukocytes to produce intracellular H2O2, a measure of oxidative function, was also significantly decreased for 7 days after injury. The paradox in this paradigm of thermal injury was the demonstration of peripheral polymorphonuclear leukocyte quantitative increases with concurrent significant qualitative impairment. Qualitative lesions included altered states of membrane depolarization and depressed oxidative capability that may individually, or collectively, reduce nonspecific immune capabilities of the host to levels that are inadequate to combat infection.

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.