Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY) 14(3), 689-706 (1992)
CONTINUOUS INFUSION OF ENDOTOXIN DEPRESSES SPLENIC BLASTOGENESIS
Herman Friedman*, Catherine Newton., Susan Press*, Thomas W. Klein', Raymond Widen*, Lester J. Vial+ and Judy A. Spitzert *Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, FL and Department of Pathology+ and Department of Physiology t Louisiana State University Medical Center, New Orleans, LA )
Continuous infusion of a sublethal dose of bacterial endotoxin into rats via an implanted osmotic pump markedly affected the blastogenic responsiveness of spleen cells to specific endotoxin as well as to the nonspecific mitogens Con A, PHA or PWM. There was also a marked alteration in lymphoid cell type and number in the spleen of the rats after continuous infusion of endotoxin,with a marked increase in plasma cell infiltration and germinal center formation. There was no significant alteration in glucocorticoid steroid levels. Control rats given saline only for a period of seven days via an implanted pump showed no or minimal effect for the first 5 days and then a delayed depression of blastogenic responses to LPS and to Con A, but this time lag contrasted markedly to the much earlier unresponsivenessof splenocytes from rats infused with endotoxin. Only a slight to moderate cellular infiltration occurred in the spleen of control rats implanted with a pump infusing saline only or an empty pump. Thus endotoxin infusion in a continuous manner via an implanted pump accounted for the early and marked suppression of responsiveness, as well as alteration in spleen size and cellularity.
INTRODUCTION Exposure of an individual to single or multiple doses of a bacterial endotoxin markedly affects many physiological and metabolic functions as well as immunologic activities (1-6). Usually endotoxins, either crude lipopolysaccharide (LPS) or purified components, are injected into experimental animals or man as a single bolus at various times prior to, during, or after monitoring for either physiological or immunological responsiveness. Recently, a model system has been developed to study LPS effects on physiological function and metabolism based on continuous infusion of bacterial endotoxin over a period of days using
689
Copyright 0 1992 by Marcel Dekker, Inc.
FRIEDMAN ET AL.
690
an osmotic pump implanted into rats (7-10). The pump is implanted subcutaneously in the dorsal region of the rat for a period up to a week and osmotic pressure results first in a slow infusion of saline and then a non-lethal dose of endotoxin into a jugular vein through a Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
catheter. Alterations of metabolic functions and some physiological parameters, such as blood pressure and in vitro myocardial activity, occurred in the rats infused with endotoxin via the pump (7-10). Only minimal effects occurred in control animals with a pump containing saline. Furthermore, the rats given continuous infusion of endotoxin showed a reversal to normal metabolic and physiological functions within a period of several days, even though the endotoxin was still present systemically as measured by tests to quantitate the presence of circulating endotoxin.
It was of interest to determine whether endotoxin administered in a continuous manner by the osmotic pump would also affect immunologic parameters. Injection of single or multiple doses of endotoxin into experimental animals like as rodents by a bolus in a parental site subcutaneously or intravenously results in either enhanced or depressed immune responses, depending upon the dosage and timing of the endotoxin administered relative to the immune parameter being assessed (6). Thus it was surprising to note that when LPS was given continuously via an implanted pump only suppressed responses occurred as determined by blastogenic assay of splenocytes against several stimulators, including the endotoxin itself (11). The present study is an extension of the earlier preliminary study and detailed the nature and possible mechanisms involved.
METHODS AND MATERIALS Animals - Male Sprague Dawley rats weighing approximately 300-350 gms each were
used for these studies. The animals were purchased from Charles River Co., Cambridge, MA, and kept in groups of two in plastic rat cages. They were fed commercial rat food and
water ad libitum. All animals were acclimated to the laboratory for one week before use.
ENDOTOXIN AND BLASTOGENESIS
691
Osmotic Puma - Alzet 2 ml osmotic pumps were purchased from the Alza Corp., (Palo Alto, CA). Each pump measured 1.4 x 5.1 cm and weighed approximately 15.0 gms when
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
empty. The pumps had a reservoir volume of 2 ml and delivered the content at a normal rate of 10 pl per hr for up to 7 days. Endotoxin - Esherichiae cofi endotoxin (026 B8 lipopolysaccharide B) was purchased from Difco Laboratories (Detroit, MI). The endotoxin dose which resulted in noticeable morbidity but no letha!ity was 0.1 mg per 100 g body weight per day. The level of endotoxin in plasma or in the saline was determined by a quantiative Limulus lysate colorimetric assay which had a sensitivity of 0.01 ng/ml (9). Osmotic PumD ImDlantation - Each animal was operated on as described previously
and an osmotic pump placed under the dorsal skin (7-10). The pump was connected through a prefabricated capillary tubing coil into the right jugular vein. All animals given fluids through the osmotic pump received saline for the first 42 hr. Those animals receiving endotoxin had the LPS administered from the same pump from 42 hr up to 7 days postoperatively. Other control rats in the same experiments were implanted with an empty Alzet pump. Infusion bv a Swivel Cannula Tether - For some experiments the animals were tethered to a peristaltic pump outside the cage and a plastic tubing inserted into the jugular vein. A small animal Series 375 infusion swivel (Instech Laboratories, Horsham, PA) with a
transmitting coil spring was used. A lightweight stainless steel anchor button was fastened
to the scapular region of the rat. The cannula passed through the button stalk into the spring tube and up to the swivel mounted above the cage. The animals could move freely about their cages.
Blastogenic responsiveness of the spleen cells of these rats was
determined 5 or 10 days later as in previous experiments. SDleen Histology - The spleen was obtained at various times after pump implantation from experimental as well as from control rats. Splenic weight was determined as well as total body weight. Each spleen was cut in two halves transversely and one half placed in formalin and histologically examined after staining with eosin and hematoxylin.
FRIEUMAN ET AL.
692
Blastogenic Assay - Dispersed spleen cells were prepared from one half of each spleen using RPMI 1640 medium containing 10% fetal calf serum and antibiotics (12). The cell suspensions were washed several times and resuspended to a concentration of 1 x lo6viable Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
nucleated spleen cells per ml as determined by trypan blue stain technique with a hemocytometer. To each well of 96 well microtiter plates (Costar) were added 2 x 16 spleen cells in 0.1 ml sterile complete medium. Medium was added as a control to some wells. Other wells received either E. coli LPS at a dose of 10 pg/rnl, Concanavalin A (Con A), Pokeweed mitogen (PWM) or Phytohemagglutinin (PHA) each at a dose of 5 pg/ml, and each cell suspension tested in triplicate. The plates were incubated for 48 hr at 37" in an atmosphere of 95% air, 5% CO,. The cells were then pulsed with 0.5 uCi 3H-thymidine and the plates further incubated for 18 hr at 37°C. The amount of radiolabel taken up into the cells was then determined by collecting cells on glass fiber filters as described previously (12). The delta cpm was calculated as amount of radioactivity (cpm) taken up into the
stimulated cultures minus the amount of radioactivity (cpm) of control cultures without stimulator. In all cases each experimental point was based on results for spleen cells from at least 3-4 animals and each experiment was repeated several times. Corndement Level Determination - Complement levels were determined in the serum of rats implanted with an osmotic pump dispensing either endotoxin or, as a control, saline. Complement levels were also determined in the serum of control rats without an implanted pump or rats with an empty pump. Complement levels were determined by preparing twofold serial dilutions of serum in microtiter plates, to which were added sheep erythrocytes coated with anti-sheep red blood cells (13). The hemolysin level was titrated with optimally sensitized erythrocytes for lysis by complement.
The end point was calculated by
determining the degree of unlysed red blood cells per well, as compared to microwells containing red cells incubated in normal serum alone or saline. The titer was calculated as the dilution of serum which resulted in 2 + lysis of the red blood cells on a scale of 0 (no lysis) to 4 t (complete lysis).
ENDOTOXIN AND BLASTOGENESIS
693
EXPERIMENTAL RESULTS
Blastogenic ResDonses to LPS - Normal rat spleen cells readily responded by thymidine uptake after stimulation with endotoxin in vitro (Table 1, Column 7). During the first two Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
days after implantation of a pump and infusion of saline the expected responses to endotoxin occurred in spleen cell cultures from pump-implanted rats as compared to control animals. However, when endotoxin was administered via the pump starting at 42 hr there was a rapid and continuous depression of blastogenic responsiveness. This became evident one day after start of administration of the endotoxin (approximately 2 days after implantation of the pump). Blastogenic responsiveness was markedly depressed by the 3rd to 5th day after endotoxin administration via the pump (Table 1, columns 3 and 4 and Fig 1). Rats given endotoxin for a total of approximately 5 days (42 hr to 7 days after pump
implantation) continued to show markedly depressed blastogenic responsiveness. After a rest period of 3 days, the spleen cells of these rats still were inactive and evinced unresponsiveness to the endotoxin. In contrast, spleen cells from positive control rats given
a single bolus injection of 0.5 ml of the same endotoxin i.v. at a dose of 10 or 100 pg showed marked blastogenesis to 10 pg LPS 1-5 days thereafter, with the peak response at 2 days (data not shown), exactly as reported previously (12).
Blastogenic Responses to Mitogens - Rats given endotoxin via the osmotic pump and showing a depressed response to the LPS also showed a marked suppression of responsiveness to the nonspecific T cell mitogens, Con A and PHA, as well as to a mitogen which affects both T and B cells (PWM). Spleen cells from control rats without an implanted pump or without administration of endotoxin showed an excellent response to these mitogens, as did spleen cells from rats during the first days after administration of saline only via the osmotic pump (Tables 2 and 3). In contrast, spleen cells from pump implanted rats given endotoxin for one or two days or longer showed a continuing and marked suppression of blastogenic responsiveness to mitogens (Tables 2 and 3). Ten days after implantation of the pump and infusion of endotoxin for 5 days followed by a rest period for 3 days, there was still a marked unresponsiveness of the spleen cells to blastogenic
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
Effect of continuous infusion of endotoxin into rats by an osmotic pump on biastogenic responsiveness of spleen cells to lipopolysaccharide
LPS infused rats Blastoeenic response in vitrob
Saline infused rats Blastonem'c resDonse in vitrob
Spleen wt
(mg)
723 2 58 775 5 43 955 f67 1481f 102 2074 5 180
Medium
5,238 f 195 3,328 f 121 2,345 f 186 3,794 2 199 3,314 f436
plus LPS
12,3743 226 3,998 ? 401 2,420 f 156 3,1582337 3,243 f879
SI
Spleen wt (mg>
-
762240
5,36223,650
17,747f 1106
3.3
2.4 1.2
IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY) 14(3), 689-706 (1992)
CONTINUOUS INFUSION OF ENDOTOXIN DEPRESSES SPLENIC BLASTOGENESIS
Herman Friedman*, Catherine Newton., Susan Press*, Thomas W. Klein', Raymond Widen*, Lester J. Vial+ and Judy A. Spitzert *Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, FL and Department of Pathology+ and Department of Physiology t Louisiana State University Medical Center, New Orleans, LA )
Continuous infusion of a sublethal dose of bacterial endotoxin into rats via an implanted osmotic pump markedly affected the blastogenic responsiveness of spleen cells to specific endotoxin as well as to the nonspecific mitogens Con A, PHA or PWM. There was also a marked alteration in lymphoid cell type and number in the spleen of the rats after continuous infusion of endotoxin,with a marked increase in plasma cell infiltration and germinal center formation. There was no significant alteration in glucocorticoid steroid levels. Control rats given saline only for a period of seven days via an implanted pump showed no or minimal effect for the first 5 days and then a delayed depression of blastogenic responses to LPS and to Con A, but this time lag contrasted markedly to the much earlier unresponsivenessof splenocytes from rats infused with endotoxin. Only a slight to moderate cellular infiltration occurred in the spleen of control rats implanted with a pump infusing saline only or an empty pump. Thus endotoxin infusion in a continuous manner via an implanted pump accounted for the early and marked suppression of responsiveness, as well as alteration in spleen size and cellularity.
INTRODUCTION Exposure of an individual to single or multiple doses of a bacterial endotoxin markedly affects many physiological and metabolic functions as well as immunologic activities (1-6). Usually endotoxins, either crude lipopolysaccharide (LPS) or purified components, are injected into experimental animals or man as a single bolus at various times prior to, during, or after monitoring for either physiological or immunological responsiveness. Recently, a model system has been developed to study LPS effects on physiological function and metabolism based on continuous infusion of bacterial endotoxin over a period of days using
689
Copyright 0 1992 by Marcel Dekker, Inc.
FRIEDMAN ET AL.
690
an osmotic pump implanted into rats (7-10). The pump is implanted subcutaneously in the dorsal region of the rat for a period up to a week and osmotic pressure results first in a slow infusion of saline and then a non-lethal dose of endotoxin into a jugular vein through a Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
catheter. Alterations of metabolic functions and some physiological parameters, such as blood pressure and in vitro myocardial activity, occurred in the rats infused with endotoxin via the pump (7-10). Only minimal effects occurred in control animals with a pump containing saline. Furthermore, the rats given continuous infusion of endotoxin showed a reversal to normal metabolic and physiological functions within a period of several days, even though the endotoxin was still present systemically as measured by tests to quantitate the presence of circulating endotoxin.
It was of interest to determine whether endotoxin administered in a continuous manner by the osmotic pump would also affect immunologic parameters. Injection of single or multiple doses of endotoxin into experimental animals like as rodents by a bolus in a parental site subcutaneously or intravenously results in either enhanced or depressed immune responses, depending upon the dosage and timing of the endotoxin administered relative to the immune parameter being assessed (6). Thus it was surprising to note that when LPS was given continuously via an implanted pump only suppressed responses occurred as determined by blastogenic assay of splenocytes against several stimulators, including the endotoxin itself (11). The present study is an extension of the earlier preliminary study and detailed the nature and possible mechanisms involved.
METHODS AND MATERIALS Animals - Male Sprague Dawley rats weighing approximately 300-350 gms each were
used for these studies. The animals were purchased from Charles River Co., Cambridge, MA, and kept in groups of two in plastic rat cages. They were fed commercial rat food and
water ad libitum. All animals were acclimated to the laboratory for one week before use.
ENDOTOXIN AND BLASTOGENESIS
691
Osmotic Puma - Alzet 2 ml osmotic pumps were purchased from the Alza Corp., (Palo Alto, CA). Each pump measured 1.4 x 5.1 cm and weighed approximately 15.0 gms when
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
empty. The pumps had a reservoir volume of 2 ml and delivered the content at a normal rate of 10 pl per hr for up to 7 days. Endotoxin - Esherichiae cofi endotoxin (026 B8 lipopolysaccharide B) was purchased from Difco Laboratories (Detroit, MI). The endotoxin dose which resulted in noticeable morbidity but no letha!ity was 0.1 mg per 100 g body weight per day. The level of endotoxin in plasma or in the saline was determined by a quantiative Limulus lysate colorimetric assay which had a sensitivity of 0.01 ng/ml (9). Osmotic PumD ImDlantation - Each animal was operated on as described previously
and an osmotic pump placed under the dorsal skin (7-10). The pump was connected through a prefabricated capillary tubing coil into the right jugular vein. All animals given fluids through the osmotic pump received saline for the first 42 hr. Those animals receiving endotoxin had the LPS administered from the same pump from 42 hr up to 7 days postoperatively. Other control rats in the same experiments were implanted with an empty Alzet pump. Infusion bv a Swivel Cannula Tether - For some experiments the animals were tethered to a peristaltic pump outside the cage and a plastic tubing inserted into the jugular vein. A small animal Series 375 infusion swivel (Instech Laboratories, Horsham, PA) with a
transmitting coil spring was used. A lightweight stainless steel anchor button was fastened
to the scapular region of the rat. The cannula passed through the button stalk into the spring tube and up to the swivel mounted above the cage. The animals could move freely about their cages.
Blastogenic responsiveness of the spleen cells of these rats was
determined 5 or 10 days later as in previous experiments. SDleen Histology - The spleen was obtained at various times after pump implantation from experimental as well as from control rats. Splenic weight was determined as well as total body weight. Each spleen was cut in two halves transversely and one half placed in formalin and histologically examined after staining with eosin and hematoxylin.
FRIEUMAN ET AL.
692
Blastogenic Assay - Dispersed spleen cells were prepared from one half of each spleen using RPMI 1640 medium containing 10% fetal calf serum and antibiotics (12). The cell suspensions were washed several times and resuspended to a concentration of 1 x lo6viable Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
nucleated spleen cells per ml as determined by trypan blue stain technique with a hemocytometer. To each well of 96 well microtiter plates (Costar) were added 2 x 16 spleen cells in 0.1 ml sterile complete medium. Medium was added as a control to some wells. Other wells received either E. coli LPS at a dose of 10 pg/rnl, Concanavalin A (Con A), Pokeweed mitogen (PWM) or Phytohemagglutinin (PHA) each at a dose of 5 pg/ml, and each cell suspension tested in triplicate. The plates were incubated for 48 hr at 37" in an atmosphere of 95% air, 5% CO,. The cells were then pulsed with 0.5 uCi 3H-thymidine and the plates further incubated for 18 hr at 37°C. The amount of radiolabel taken up into the cells was then determined by collecting cells on glass fiber filters as described previously (12). The delta cpm was calculated as amount of radioactivity (cpm) taken up into the
stimulated cultures minus the amount of radioactivity (cpm) of control cultures without stimulator. In all cases each experimental point was based on results for spleen cells from at least 3-4 animals and each experiment was repeated several times. Corndement Level Determination - Complement levels were determined in the serum of rats implanted with an osmotic pump dispensing either endotoxin or, as a control, saline. Complement levels were also determined in the serum of control rats without an implanted pump or rats with an empty pump. Complement levels were determined by preparing twofold serial dilutions of serum in microtiter plates, to which were added sheep erythrocytes coated with anti-sheep red blood cells (13). The hemolysin level was titrated with optimally sensitized erythrocytes for lysis by complement.
The end point was calculated by
determining the degree of unlysed red blood cells per well, as compared to microwells containing red cells incubated in normal serum alone or saline. The titer was calculated as the dilution of serum which resulted in 2 + lysis of the red blood cells on a scale of 0 (no lysis) to 4 t (complete lysis).
ENDOTOXIN AND BLASTOGENESIS
693
EXPERIMENTAL RESULTS
Blastogenic ResDonses to LPS - Normal rat spleen cells readily responded by thymidine uptake after stimulation with endotoxin in vitro (Table 1, Column 7). During the first two Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
days after implantation of a pump and infusion of saline the expected responses to endotoxin occurred in spleen cell cultures from pump-implanted rats as compared to control animals. However, when endotoxin was administered via the pump starting at 42 hr there was a rapid and continuous depression of blastogenic responsiveness. This became evident one day after start of administration of the endotoxin (approximately 2 days after implantation of the pump). Blastogenic responsiveness was markedly depressed by the 3rd to 5th day after endotoxin administration via the pump (Table 1, columns 3 and 4 and Fig 1). Rats given endotoxin for a total of approximately 5 days (42 hr to 7 days after pump
implantation) continued to show markedly depressed blastogenic responsiveness. After a rest period of 3 days, the spleen cells of these rats still were inactive and evinced unresponsiveness to the endotoxin. In contrast, spleen cells from positive control rats given
a single bolus injection of 0.5 ml of the same endotoxin i.v. at a dose of 10 or 100 pg showed marked blastogenesis to 10 pg LPS 1-5 days thereafter, with the peak response at 2 days (data not shown), exactly as reported previously (12).
Blastogenic Responses to Mitogens - Rats given endotoxin via the osmotic pump and showing a depressed response to the LPS also showed a marked suppression of responsiveness to the nonspecific T cell mitogens, Con A and PHA, as well as to a mitogen which affects both T and B cells (PWM). Spleen cells from control rats without an implanted pump or without administration of endotoxin showed an excellent response to these mitogens, as did spleen cells from rats during the first days after administration of saline only via the osmotic pump (Tables 2 and 3). In contrast, spleen cells from pump implanted rats given endotoxin for one or two days or longer showed a continuing and marked suppression of blastogenic responsiveness to mitogens (Tables 2 and 3). Ten days after implantation of the pump and infusion of endotoxin for 5 days followed by a rest period for 3 days, there was still a marked unresponsiveness of the spleen cells to blastogenic
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by UB Wuerzburg on 12/10/14 For personal use only.
Effect of continuous infusion of endotoxin into rats by an osmotic pump on biastogenic responsiveness of spleen cells to lipopolysaccharide
LPS infused rats Blastoeenic response in vitrob
Saline infused rats Blastonem'c resDonse in vitrob
Spleen wt
(mg)
723 2 58 775 5 43 955 f67 1481f 102 2074 5 180
Medium
5,238 f 195 3,328 f 121 2,345 f 186 3,794 2 199 3,314 f436
plus LPS
12,3743 226 3,998 ? 401 2,420 f 156 3,1582337 3,243 f879
SI
Spleen wt (mg>
-
762240
5,36223,650
17,747f 1106
3.3
2.4 1.2
