Clin. exp. Immunol. (1992) 90, 497-502
Haemorrhage-induced alterations in function and cytokine production of T cells and T cell subpopulations E. ABRAHAM & Y.-H. CHANG* Departments of Medicine and *Pharmacology, UCLA School of Medicine, Los Angeles, CA, USA
(Accepted for publication 11 September 1992)
SUMMARY Haemorrhage produces alterations in macrophage, T and B cell function. In order to better define the mechanism for the effects of blood loss on immune response, we examined function of and cytokine production by purified T cells, CD4+ and CD8+ subpopulations after blood loss. Whereas T and CD4+ cells from control, unhaemorrhaged animals produced no alteration in proliferation when added to cultures of mitogen-stimulated splenocytes from normal mice, proliferation was decreased when T or CD4+ cells from haemorrhaged mice were included. The addition of CD8+ cells from haemorrhaged animals to mitogen-stimulated cultures reduced proliferation by approximately 50% more than that found when CD8+ cells from control, unhaemorrhaged animals were included. Supernatants of mitogen-stimulated splenocytes from haemorrhaged mice contained significantly less IL-2 and interferon-gamma (IFN-y) than did those from control, unhaemorrhaged mice. CD4+ populations from haemorrhaged mice produced significantly more IL- IO, and significantly less IFNy, than did CD4+ cells from control, unhaemorrhaged mice. There were no significant differences in IL-2, IL-4, IL-10 or IFN-y production by CD8+ cells from haemorrhaged or control mice. The present experiments demonstrate that haemorrhage affects both CD4+ and CD8+ T cell subsets. In particular, haemorrhage appeared to activate CD4+, Th2 cells, with concomitant suppression of the Thl subpopulation. These results provide a mechanism which may contribute to the alterations in cytokine production previously described to occur following blood loss. Keywords haemorrhage T cells CD4+ cells CD8+ cells cytokines IL-2 IL-4 IL-10 interferon-gamma Thl Th2
INTRODUCTION
plasma cells producing antibody to bacterial polysaccharide antigens [13]. Macrophages also appear to be affected by haemorrhage, with decreased expression of MHC class II (Ia) and Fc receptors [9,14]. In addition, macrophage antigenpresenting ability is reduced following blood loss [9]. Mortality from normally non-lethal bacterial infection is increased following haemorrhage, even if resuscitated [3,15], suggesting that host defence functions are adversely impacted by the alterations in immune function which follow blood loss. Cytokines produced by CD4+ and CD8+ T cells are capable of affecting, in suppressive and/or stimulatory fashion, the function of other T cells, macrophages and B cells. CD4+ cells in mice, and possibly in humans, can be divided into Th I and Th2 subsets on the basis of differential cytokine production profiles [16]. Thl cells produce IFN-y, IL-2 and tumour necrosis factorbeta (TNF-,B), while Th2 cells secrete IL-4, IL-5 and IL-10 [16,17]. Linkage of the relative activities of Thl and Th2
Widespread alterations in T and B cell function have been described following haemorrhage [1-10]. Blood loss does not result in changes in the absolute or relative numbers of T and B cell subsets (CD3+, CD4+, CD8+, Ig+ or B220+) in spleen, lymph nodes, bone marrow, thymus or lungs [5,7]. However, alterations in cytokine (IL-1, IL-2, IL-3, IL-5, IL-6, interferongamma (IFN-y)) release [4,5,10], decreases in mitogen-induced T cell proliferation [3,5], reduction in IL-2 receptor expression [5], changes in B cell clonal precursor frequencies [6-8], and decreases in bacterial antigen-specific plasma cell numbers and frequencies [6,8,1 1 ] occur after haemorrhage. In addition, blood loss appears to activate a population of CD8 + T cells, capable of diminishing in vivo alloantigen-specific cellular cytotoxicity when infused into normal animals [12]. Transfer of these haemorrhage-activated CD8+ cells to normal, unhaemorrhaged mice results in decreased numbers and frequencies of splenic
subpopulations is accomplished, in part, by Th2-produced IL10, which suppresses cytokine generation of Thl cells through a macrophage-mediated mechanism [18]. In the present studies, in order to better define the mechanisms underlying the haemorrhage-induced alterations in
Correspondence: Edward Abraham MD, Division of Pulmonary and Critical Care Medicine, Department of Medicine, UCLA Medical Centre, Los Angeles, CA 90024, USA.
497
498
E. Abraham & Y.-H. Chang
immune response, we examined function and cytokine production of purified T cells, CD4+ and CD8+ subpopulations after blood loss. These experiments demonstrate that haemorrhage affects both CD4+ and CD8+ subsets, and, among CD4+ cells, appeared to activate the Th2 subpopulation, with concomitant down-regulation of Thl cytokine production. MATERIALS AND METHODS Animals Male BALB/c mice, 8-12 weeks of age, were obtained from Jackson Laboratory (Bar Harbor, ME). The animals were kept on a 12-h light/dark cycle with free access to food and water.
Haemorrhage The model for haemorrhage and resuscitation has been described previously [19]. Briefly, mice were anaesthetized with inhaled ether after being placed into a covered beaker. Cardiac puncture was performed with a 30 G needle and 30% of the calculated blood volume (approximately 0 55 ml for a 20-g mouse) was withdrawn over a 60-s period into a heparinized syringe and stored at 37°C. The mouse then was allowed to recuperate in its cage. The total period of ether anaesthesia was less than 2 min in all cases. One hour after haemorrhage, the removed blood was reinfused into the unanaesthetized mouse through the retroorbital plexus. The mortality rate with this haemorrhage protocol is approximately 12%, with all deaths occurring over the 24 h post haemorrhage, and most deaths occurring within the 1 h post haemorrhage [19]. With this model, ether anaesthesia and cardiac puncture without blood withdrawal result in no changes in susceptibility to infection, mitogen-induced lymphocyte proliferation, IL-2R expression, phenotypic characteristics (CD3, CD4, CD8, B220, u, Ly-l expression) of B or T lymphocytes, lymphokine (IL-2, IL-3, IL5, IFN-y) release, or splenic, intestinal, or pulmonary B cell clonal precursor frequencies [4-8,15,19]. Similarly, no evidence of haemothorax, bleeding into the pericardial space, lung or cardiac contusion has been found in surviving mice with this method of haemorrhage [5]. Reagents RPMI 1640, fetal calf serum (FCS), HEPES, 2-mercaptoethanol (2-ME), L-glutamine, and antibiotics (penicillin/streptomycin) were from Sigma (St Louis, MO). Concanavalin A (Con A) was from Pharmacia (Uppsala, Sweden). Tritiated thymidine (3H-thymidine; specific activity 5 Ci/mmol) was from Amersham (Arlington Heights, IL).
Purification of T cells and T cell subsets Spleens from normal or 3-day post haemorrhage BALB/c mice were used for the isolation of CD4+ and CD8+ cells. The timepoint 3 days post haemorrhage was chosen because previous experiments [5,12] had demonstrated maximum suppression of Con A-induced proliferation, proliferation in MLR, and IL-2R expression at this time after blood loss. Spleens were teased apart in RPMI 1640, supplemented with HEPES, antibiotics, 2-ME and glutamine. The cells were washed twice with this medium, then erythrocytes lysed by a 1l-mmntreatment with Gey's solution at 4°C.The remaining cells were incubated on nylon wool columns, and the nylon wool non-adherent population treated with complement after incu-
bation either with anti-I-A alone (mouse anti-mouse anti-I-Ad, obtained from Becton Dickinson, Mountain View, CA) to obtain purified T cells, or with anti-I-A and anti-CD4 (H 12919.69) [20] or anti-CD8 (HO-2.2) [21] antibodies, to obtain purified CD8+ or CD4+ cells. In several experiments, splenocytes were depleted of CD4+ or CD8+ cells using anti-CD4 or anti-CD8 antibodies and complement, without additional treatment of cells on nylon wool columns or with anti-I-A antibodies. Purification of the resultant CD4+ and CD8+ populations was verified with flow cytometry. In each case, the final cell populations contained less than 2% I-A+, Ig+, and, if treated with anti-CD4+ or anti-CD8+, alternate T cell subset (CD4+ or CD8+) contamination. Cell cultures Culture medium was RPMI 1640 supplemented with 10 mM HEPES, 50 IM 2-ME, 20 mM L-glutamine, antibiotics and 10% FCS. Con A was added to the cultures at a concentration of 2 5 pg/ml. Microcultures were carried out in 96-well, flat-bottomed plates, with each well containing 2 x 105 cells in 0-2 ml of culture medium. Incubation conditions were in 5% CO2 at 37°C. All cultures were carried out with at least five replicates. Supernatants for assays of IL and IFN-y were collected after 24 h of culture in order to monitor cytokine production during log phase increase and to avoid possible degradation of cytokines which may occur when supernatents are collected in the plateau phase. In proliferation assays, a 4-h pulse of I yCi/ well of 3H-thymidine was used after 72 h of culture. The cells then were collected onto glass fibre filter paper with a cell harvester (Skatron, Oslo, Norway) and 3H-thymidine incorporation assayed in a scintillation counter.
IL and JFN-y assays IL-2 content of supernatants obtained from splenocyte cultures of normal and haemorrhaged animals was assessed using the cell line CTLL-2, as previously described [5]. Because these cells respond to IL-4 as well as IL-2, the anti-IL-4 MoAb 1 lBl 1 [22] (a gift of Dr D. Ando, Cetus Corporation, Emeryville, CA) was added to the cultures, and no response to exogenously provided IL-4 (a gift of Dr Paul Stevens, Amgen, Thousand Oaks, CA) was found. In the IL-2 assay, 104 CTLL-2 cells were cultured in 0-2 ml volumes containing serial two-fold dilutions of supernatants, ranging from 1:2 to 1:1024. The culture media in all cases was RPMI 1640 supplemented with 10 mm HEPES, 50 pM 2-ME, antibiotics, glutamine and 10% FCS. Serial dilutions of recombinant IL-2 (a gift from Dr Paul Stevens) also were cultured with the CTLL-2 cells to provide a standard for comparison between supernatants and assays. For each supernatant the series of dilutions was performed in triplicate. After 24 h of culture in 5% CO2 at 37°C, the cultures were pulsed for 4 h with 1 pCi/well of 3H-thymidine. Analysis of the IL-2 content of each supernatant was then determined by probit analysis, with the number of units of IL-2 in the supernatants being calculated by comparison with that determined for the recombinant control IL (initial dilution 300 World Health
Organization (WHO) U/ml). The content of IL-4 and IFN-y in the supernatants was determined using an ELISA technique with commercially available ELISA kits (IL-4 ELISA obtained from Endogen, Boston, MA, and IFN-y ELISA obtained from GIBCO BRL, Gaithersburg, MD). In the IL-4 assay, a calibration curve was
T, CD4+ and CD8+ cells following haemorrhage Table 1. The effects of haemorrhage and resuscitation on T, CD4+ and CD8+ cell function
Population added Normal splenocytes Control T cells Haemorrhage T cells Control CD4t Haemorrhage CD4t Control CD8t Haemorrhage CD8t
ct/mmn 160 124+ 10-814 166 940+ 11 486 125666 + 16.202* 151215+ 14 189 133.937 + 3122* 130-769 + 7329* 115-161+5602t
Mice (n= 5 in each group) were ether anaesthetized, then either haemorrhaged and resuscitated 1 h later (haemorrhage) or subjected only to cardiac puncture, without blood withdrawal (control). T cells and T cell subpopulations (CD4+ and CD8+) were collected 3 days later. Splenocytes (1 x l05) from normal, unmanipulated mice were placed into culture for 72 h with concanavalin A (Con A) and cell populations (I x l0J) as noted. During
the final 4 h of culture, 1 pCi of 3H-thymidine was added. Results are presented as mean + s.d. * P < 0 05 and tP < 0-01 versus cultures containing 2 x 105 normal splenocytes (normal).
constructed using 0-375 pg/ml recombinant murine IL-4 (1 pg IL-4 was equivalent to 0 02 U as determined in a standardized bioassay using the IL-4 sensitive CT.4S cell line) [23]. In the IFN-y assay, a calibration curve was constructed using 0-7000 pg/ml recombinant murine IFN-y (175 pg of the recombinant murine IFN-y is equivalent to I NIH standard unit). For IL-IO, an ELISA was performed using monoclonal rat anti-mouse IL-IO (18141D, Pharmingen, San Diego, CA) as the capture antibody, and a biotinylated rat anti-mouse IL-10 MoAb (18152D, Pharmingen) as the detecting antibody. A standard curve was prepared in the IL-10 ELISA using recombinant IL-lO, produced in a COS7 expression system (Pharmingen), with titres of 0-470 U/ml in the calibration curve (one IL-10 unit is defined as the amount which will inhibit by 50% the production of IFN-y by Thl cells (clone 12-11, 50000 cells/well, with the wells also containing 106 irradiated syngeneic (A/J) splenocytes) stimulated by culture with 50 ng/well human gamma globulin for 22 h) [24,25]. The threshold for detection of IL-10 in this ELISA was 1 U/ml. For all cytokine ELISAs, supernatants were tested in duplicate.
499
affect mitogen-induced proliferation of normal splenocytes, we performed a cell-mixing experiment where 1 x 105 total T, CD8+, or CD4+ cells from mice subjected to ether anaesthesia and cardiac puncture, but no blood withdrawal, or from haemorrhaged and resuscitated mice were added to I x 105 splenocytes, isolated from normal mice, and then cultured with Con A. The results of these experiments are shown in Table 1. Cultures containing cells from haemorrhaged animals, whether total T cells, CD8+, or CD4+ subpopulations, showed significantly decreased proliferation compared with comparable cultures containing the same T cell populations or subpopulations from mice subjected to ether anaesthesia and cardiac puncture, but without blood withdrawal (Table 1). The proliferation of cultures containing purified T cells from haemorrhaged mice was decreased by approximately 25%, as compared with that seen in cultures containing T cells from unhaemorrhaged mice (Table 1). Similarly, while the addition of purified CD4+ cells from unmanipulated mice to normal splenocytes produced no significant change in proliferation compared with cultures containing only splenocytes, the inclusion of CD4+ cells from haemorrhaged mice in these cultures resulted in 16% less proliferation. Proliferation in cultures containing purified CD8 + cells from normal mice was approximately 18% less than in cultures containing only splenocytes, whereas addition of CD8 + cells from haemorrhaged mice resulted in an almost twofold greater decrease in proliferation.
Effects of haemorrhage on cytokine generation The previous experiments demonstrated that haemorrhage affected both CD4+ and CD8+ populations in terms of their interactions with normal splenocytes in mitogen-driven proliferative cultures. In order to investigate possible mechanisms by which haemorrhage could produce alterations in T cell, CD4+ and CD8+ function, we measured levels of IL-2, IL-4, IL-IO and IFN-y in supernatants of mitogen-stimulated cultures from control, unhaemorrhaged and from haemorrhaged mice. To determine the relative effects of haemorrhage on CD4+ and CD8 + subpopulations, cytolytic antibodies were used to deplete either the CD4+ or CD8+ cells from the proliferative cultures. The results of these experiments are shown in Table 2. Significant decrease in IL-2 and IFN-y production, to approximately 40% of control levels, was found among splenocytes obtained following haemorrhage (Table 2). IL-4 generation was not significantly altered in cells isolated following blood loss, compared with that found in supernatants from control mice. No detectable IL- 10 was found in culture supernatants from control or haemorrhaged mice. Statistical analysis In cultures from haemorrhaged mice containing CD4+ cells, Data are presented as mean + s.d. for each experimental group. but no CD8+ cells, IL-10 titres were increased to approximately Comparisons between groups were performed by Student's 15 U/ml whereas levels of IL- 10 in similar cultures from control one-way t-test for differences between two groups or by using mice /were approximately 15-fold less. Production of IFN-y was experment * ~~~~fr * * ^ * * analysis of variance for examining differences orreduced by approximately 30% in the cultures from haemorwith more than two groups. A P value less than 0 05 was reudbyapoitly3%nthcluesfmheoconsidered to be significant. rhaged mice which contained CD4+, but no CD8+ cells. Approximately 20% less IL-2 and approximately 20% more IL4 was found in the CD4+ cultures from haemorrhaged mice, as compared with controls, but these differences did not achieve RESULTS statistical significance. In contrast, haemorrhage resulted in no significant differences in IL-2, IL-4, IL-10 or IFN-y production Effects of haemorrhage on T cell subsets among cultures containing CD8+ cells, but depleted of CD4+ In order to determine the effects of haemorrhage and resuscicells. tation on the functional abilities of T cells and T cell subsets to
500
E. Abraham & Y.-H. Chang Table 2. The effects of haemorrhage and resuscitation on cytokine production
Population Total CD4+ CD8+
IL-10 (U/ml)
IL-4 (pg/ml)
IFN-y (pg/ml)
IL-2 (U/ml)
C
H
C
H
C
H
C
H
553+ 144 191+116 45+27
232+ 118* 153+102 38+3
2981 + 1203 481+90 478+255
1216+500*
49+ 19 21+11 5+4
44+8
Haemorrhage-induced alterations in function and cytokine production of T cells and T cell subpopulations E. ABRAHAM & Y.-H. CHANG* Departments of Medicine and *Pharmacology, UCLA School of Medicine, Los Angeles, CA, USA
(Accepted for publication 11 September 1992)
SUMMARY Haemorrhage produces alterations in macrophage, T and B cell function. In order to better define the mechanism for the effects of blood loss on immune response, we examined function of and cytokine production by purified T cells, CD4+ and CD8+ subpopulations after blood loss. Whereas T and CD4+ cells from control, unhaemorrhaged animals produced no alteration in proliferation when added to cultures of mitogen-stimulated splenocytes from normal mice, proliferation was decreased when T or CD4+ cells from haemorrhaged mice were included. The addition of CD8+ cells from haemorrhaged animals to mitogen-stimulated cultures reduced proliferation by approximately 50% more than that found when CD8+ cells from control, unhaemorrhaged animals were included. Supernatants of mitogen-stimulated splenocytes from haemorrhaged mice contained significantly less IL-2 and interferon-gamma (IFN-y) than did those from control, unhaemorrhaged mice. CD4+ populations from haemorrhaged mice produced significantly more IL- IO, and significantly less IFNy, than did CD4+ cells from control, unhaemorrhaged mice. There were no significant differences in IL-2, IL-4, IL-10 or IFN-y production by CD8+ cells from haemorrhaged or control mice. The present experiments demonstrate that haemorrhage affects both CD4+ and CD8+ T cell subsets. In particular, haemorrhage appeared to activate CD4+, Th2 cells, with concomitant suppression of the Thl subpopulation. These results provide a mechanism which may contribute to the alterations in cytokine production previously described to occur following blood loss. Keywords haemorrhage T cells CD4+ cells CD8+ cells cytokines IL-2 IL-4 IL-10 interferon-gamma Thl Th2
INTRODUCTION
plasma cells producing antibody to bacterial polysaccharide antigens [13]. Macrophages also appear to be affected by haemorrhage, with decreased expression of MHC class II (Ia) and Fc receptors [9,14]. In addition, macrophage antigenpresenting ability is reduced following blood loss [9]. Mortality from normally non-lethal bacterial infection is increased following haemorrhage, even if resuscitated [3,15], suggesting that host defence functions are adversely impacted by the alterations in immune function which follow blood loss. Cytokines produced by CD4+ and CD8+ T cells are capable of affecting, in suppressive and/or stimulatory fashion, the function of other T cells, macrophages and B cells. CD4+ cells in mice, and possibly in humans, can be divided into Th I and Th2 subsets on the basis of differential cytokine production profiles [16]. Thl cells produce IFN-y, IL-2 and tumour necrosis factorbeta (TNF-,B), while Th2 cells secrete IL-4, IL-5 and IL-10 [16,17]. Linkage of the relative activities of Thl and Th2
Widespread alterations in T and B cell function have been described following haemorrhage [1-10]. Blood loss does not result in changes in the absolute or relative numbers of T and B cell subsets (CD3+, CD4+, CD8+, Ig+ or B220+) in spleen, lymph nodes, bone marrow, thymus or lungs [5,7]. However, alterations in cytokine (IL-1, IL-2, IL-3, IL-5, IL-6, interferongamma (IFN-y)) release [4,5,10], decreases in mitogen-induced T cell proliferation [3,5], reduction in IL-2 receptor expression [5], changes in B cell clonal precursor frequencies [6-8], and decreases in bacterial antigen-specific plasma cell numbers and frequencies [6,8,1 1 ] occur after haemorrhage. In addition, blood loss appears to activate a population of CD8 + T cells, capable of diminishing in vivo alloantigen-specific cellular cytotoxicity when infused into normal animals [12]. Transfer of these haemorrhage-activated CD8+ cells to normal, unhaemorrhaged mice results in decreased numbers and frequencies of splenic
subpopulations is accomplished, in part, by Th2-produced IL10, which suppresses cytokine generation of Thl cells through a macrophage-mediated mechanism [18]. In the present studies, in order to better define the mechanisms underlying the haemorrhage-induced alterations in
Correspondence: Edward Abraham MD, Division of Pulmonary and Critical Care Medicine, Department of Medicine, UCLA Medical Centre, Los Angeles, CA 90024, USA.
497
498
E. Abraham & Y.-H. Chang
immune response, we examined function and cytokine production of purified T cells, CD4+ and CD8+ subpopulations after blood loss. These experiments demonstrate that haemorrhage affects both CD4+ and CD8+ subsets, and, among CD4+ cells, appeared to activate the Th2 subpopulation, with concomitant down-regulation of Thl cytokine production. MATERIALS AND METHODS Animals Male BALB/c mice, 8-12 weeks of age, were obtained from Jackson Laboratory (Bar Harbor, ME). The animals were kept on a 12-h light/dark cycle with free access to food and water.
Haemorrhage The model for haemorrhage and resuscitation has been described previously [19]. Briefly, mice were anaesthetized with inhaled ether after being placed into a covered beaker. Cardiac puncture was performed with a 30 G needle and 30% of the calculated blood volume (approximately 0 55 ml for a 20-g mouse) was withdrawn over a 60-s period into a heparinized syringe and stored at 37°C. The mouse then was allowed to recuperate in its cage. The total period of ether anaesthesia was less than 2 min in all cases. One hour after haemorrhage, the removed blood was reinfused into the unanaesthetized mouse through the retroorbital plexus. The mortality rate with this haemorrhage protocol is approximately 12%, with all deaths occurring over the 24 h post haemorrhage, and most deaths occurring within the 1 h post haemorrhage [19]. With this model, ether anaesthesia and cardiac puncture without blood withdrawal result in no changes in susceptibility to infection, mitogen-induced lymphocyte proliferation, IL-2R expression, phenotypic characteristics (CD3, CD4, CD8, B220, u, Ly-l expression) of B or T lymphocytes, lymphokine (IL-2, IL-3, IL5, IFN-y) release, or splenic, intestinal, or pulmonary B cell clonal precursor frequencies [4-8,15,19]. Similarly, no evidence of haemothorax, bleeding into the pericardial space, lung or cardiac contusion has been found in surviving mice with this method of haemorrhage [5]. Reagents RPMI 1640, fetal calf serum (FCS), HEPES, 2-mercaptoethanol (2-ME), L-glutamine, and antibiotics (penicillin/streptomycin) were from Sigma (St Louis, MO). Concanavalin A (Con A) was from Pharmacia (Uppsala, Sweden). Tritiated thymidine (3H-thymidine; specific activity 5 Ci/mmol) was from Amersham (Arlington Heights, IL).
Purification of T cells and T cell subsets Spleens from normal or 3-day post haemorrhage BALB/c mice were used for the isolation of CD4+ and CD8+ cells. The timepoint 3 days post haemorrhage was chosen because previous experiments [5,12] had demonstrated maximum suppression of Con A-induced proliferation, proliferation in MLR, and IL-2R expression at this time after blood loss. Spleens were teased apart in RPMI 1640, supplemented with HEPES, antibiotics, 2-ME and glutamine. The cells were washed twice with this medium, then erythrocytes lysed by a 1l-mmntreatment with Gey's solution at 4°C.The remaining cells were incubated on nylon wool columns, and the nylon wool non-adherent population treated with complement after incu-
bation either with anti-I-A alone (mouse anti-mouse anti-I-Ad, obtained from Becton Dickinson, Mountain View, CA) to obtain purified T cells, or with anti-I-A and anti-CD4 (H 12919.69) [20] or anti-CD8 (HO-2.2) [21] antibodies, to obtain purified CD8+ or CD4+ cells. In several experiments, splenocytes were depleted of CD4+ or CD8+ cells using anti-CD4 or anti-CD8 antibodies and complement, without additional treatment of cells on nylon wool columns or with anti-I-A antibodies. Purification of the resultant CD4+ and CD8+ populations was verified with flow cytometry. In each case, the final cell populations contained less than 2% I-A+, Ig+, and, if treated with anti-CD4+ or anti-CD8+, alternate T cell subset (CD4+ or CD8+) contamination. Cell cultures Culture medium was RPMI 1640 supplemented with 10 mM HEPES, 50 IM 2-ME, 20 mM L-glutamine, antibiotics and 10% FCS. Con A was added to the cultures at a concentration of 2 5 pg/ml. Microcultures were carried out in 96-well, flat-bottomed plates, with each well containing 2 x 105 cells in 0-2 ml of culture medium. Incubation conditions were in 5% CO2 at 37°C. All cultures were carried out with at least five replicates. Supernatants for assays of IL and IFN-y were collected after 24 h of culture in order to monitor cytokine production during log phase increase and to avoid possible degradation of cytokines which may occur when supernatents are collected in the plateau phase. In proliferation assays, a 4-h pulse of I yCi/ well of 3H-thymidine was used after 72 h of culture. The cells then were collected onto glass fibre filter paper with a cell harvester (Skatron, Oslo, Norway) and 3H-thymidine incorporation assayed in a scintillation counter.
IL and JFN-y assays IL-2 content of supernatants obtained from splenocyte cultures of normal and haemorrhaged animals was assessed using the cell line CTLL-2, as previously described [5]. Because these cells respond to IL-4 as well as IL-2, the anti-IL-4 MoAb 1 lBl 1 [22] (a gift of Dr D. Ando, Cetus Corporation, Emeryville, CA) was added to the cultures, and no response to exogenously provided IL-4 (a gift of Dr Paul Stevens, Amgen, Thousand Oaks, CA) was found. In the IL-2 assay, 104 CTLL-2 cells were cultured in 0-2 ml volumes containing serial two-fold dilutions of supernatants, ranging from 1:2 to 1:1024. The culture media in all cases was RPMI 1640 supplemented with 10 mm HEPES, 50 pM 2-ME, antibiotics, glutamine and 10% FCS. Serial dilutions of recombinant IL-2 (a gift from Dr Paul Stevens) also were cultured with the CTLL-2 cells to provide a standard for comparison between supernatants and assays. For each supernatant the series of dilutions was performed in triplicate. After 24 h of culture in 5% CO2 at 37°C, the cultures were pulsed for 4 h with 1 pCi/well of 3H-thymidine. Analysis of the IL-2 content of each supernatant was then determined by probit analysis, with the number of units of IL-2 in the supernatants being calculated by comparison with that determined for the recombinant control IL (initial dilution 300 World Health
Organization (WHO) U/ml). The content of IL-4 and IFN-y in the supernatants was determined using an ELISA technique with commercially available ELISA kits (IL-4 ELISA obtained from Endogen, Boston, MA, and IFN-y ELISA obtained from GIBCO BRL, Gaithersburg, MD). In the IL-4 assay, a calibration curve was
T, CD4+ and CD8+ cells following haemorrhage Table 1. The effects of haemorrhage and resuscitation on T, CD4+ and CD8+ cell function
Population added Normal splenocytes Control T cells Haemorrhage T cells Control CD4t Haemorrhage CD4t Control CD8t Haemorrhage CD8t
ct/mmn 160 124+ 10-814 166 940+ 11 486 125666 + 16.202* 151215+ 14 189 133.937 + 3122* 130-769 + 7329* 115-161+5602t
Mice (n= 5 in each group) were ether anaesthetized, then either haemorrhaged and resuscitated 1 h later (haemorrhage) or subjected only to cardiac puncture, without blood withdrawal (control). T cells and T cell subpopulations (CD4+ and CD8+) were collected 3 days later. Splenocytes (1 x l05) from normal, unmanipulated mice were placed into culture for 72 h with concanavalin A (Con A) and cell populations (I x l0J) as noted. During
the final 4 h of culture, 1 pCi of 3H-thymidine was added. Results are presented as mean + s.d. * P < 0 05 and tP < 0-01 versus cultures containing 2 x 105 normal splenocytes (normal).
constructed using 0-375 pg/ml recombinant murine IL-4 (1 pg IL-4 was equivalent to 0 02 U as determined in a standardized bioassay using the IL-4 sensitive CT.4S cell line) [23]. In the IFN-y assay, a calibration curve was constructed using 0-7000 pg/ml recombinant murine IFN-y (175 pg of the recombinant murine IFN-y is equivalent to I NIH standard unit). For IL-IO, an ELISA was performed using monoclonal rat anti-mouse IL-IO (18141D, Pharmingen, San Diego, CA) as the capture antibody, and a biotinylated rat anti-mouse IL-10 MoAb (18152D, Pharmingen) as the detecting antibody. A standard curve was prepared in the IL-10 ELISA using recombinant IL-lO, produced in a COS7 expression system (Pharmingen), with titres of 0-470 U/ml in the calibration curve (one IL-10 unit is defined as the amount which will inhibit by 50% the production of IFN-y by Thl cells (clone 12-11, 50000 cells/well, with the wells also containing 106 irradiated syngeneic (A/J) splenocytes) stimulated by culture with 50 ng/well human gamma globulin for 22 h) [24,25]. The threshold for detection of IL-10 in this ELISA was 1 U/ml. For all cytokine ELISAs, supernatants were tested in duplicate.
499
affect mitogen-induced proliferation of normal splenocytes, we performed a cell-mixing experiment where 1 x 105 total T, CD8+, or CD4+ cells from mice subjected to ether anaesthesia and cardiac puncture, but no blood withdrawal, or from haemorrhaged and resuscitated mice were added to I x 105 splenocytes, isolated from normal mice, and then cultured with Con A. The results of these experiments are shown in Table 1. Cultures containing cells from haemorrhaged animals, whether total T cells, CD8+, or CD4+ subpopulations, showed significantly decreased proliferation compared with comparable cultures containing the same T cell populations or subpopulations from mice subjected to ether anaesthesia and cardiac puncture, but without blood withdrawal (Table 1). The proliferation of cultures containing purified T cells from haemorrhaged mice was decreased by approximately 25%, as compared with that seen in cultures containing T cells from unhaemorrhaged mice (Table 1). Similarly, while the addition of purified CD4+ cells from unmanipulated mice to normal splenocytes produced no significant change in proliferation compared with cultures containing only splenocytes, the inclusion of CD4+ cells from haemorrhaged mice in these cultures resulted in 16% less proliferation. Proliferation in cultures containing purified CD8 + cells from normal mice was approximately 18% less than in cultures containing only splenocytes, whereas addition of CD8 + cells from haemorrhaged mice resulted in an almost twofold greater decrease in proliferation.
Effects of haemorrhage on cytokine generation The previous experiments demonstrated that haemorrhage affected both CD4+ and CD8+ populations in terms of their interactions with normal splenocytes in mitogen-driven proliferative cultures. In order to investigate possible mechanisms by which haemorrhage could produce alterations in T cell, CD4+ and CD8+ function, we measured levels of IL-2, IL-4, IL-IO and IFN-y in supernatants of mitogen-stimulated cultures from control, unhaemorrhaged and from haemorrhaged mice. To determine the relative effects of haemorrhage on CD4+ and CD8 + subpopulations, cytolytic antibodies were used to deplete either the CD4+ or CD8+ cells from the proliferative cultures. The results of these experiments are shown in Table 2. Significant decrease in IL-2 and IFN-y production, to approximately 40% of control levels, was found among splenocytes obtained following haemorrhage (Table 2). IL-4 generation was not significantly altered in cells isolated following blood loss, compared with that found in supernatants from control mice. No detectable IL- 10 was found in culture supernatants from control or haemorrhaged mice. Statistical analysis In cultures from haemorrhaged mice containing CD4+ cells, Data are presented as mean + s.d. for each experimental group. but no CD8+ cells, IL-10 titres were increased to approximately Comparisons between groups were performed by Student's 15 U/ml whereas levels of IL- 10 in similar cultures from control one-way t-test for differences between two groups or by using mice /were approximately 15-fold less. Production of IFN-y was experment * ~~~~fr * * ^ * * analysis of variance for examining differences orreduced by approximately 30% in the cultures from haemorwith more than two groups. A P value less than 0 05 was reudbyapoitly3%nthcluesfmheoconsidered to be significant. rhaged mice which contained CD4+, but no CD8+ cells. Approximately 20% less IL-2 and approximately 20% more IL4 was found in the CD4+ cultures from haemorrhaged mice, as compared with controls, but these differences did not achieve RESULTS statistical significance. In contrast, haemorrhage resulted in no significant differences in IL-2, IL-4, IL-10 or IFN-y production Effects of haemorrhage on T cell subsets among cultures containing CD8+ cells, but depleted of CD4+ In order to determine the effects of haemorrhage and resuscicells. tation on the functional abilities of T cells and T cell subsets to
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E. Abraham & Y.-H. Chang Table 2. The effects of haemorrhage and resuscitation on cytokine production
Population Total CD4+ CD8+
IL-10 (U/ml)
IL-4 (pg/ml)
IFN-y (pg/ml)
IL-2 (U/ml)
C
H
C
H
C
H
C
H
553+ 144 191+116 45+27
232+ 118* 153+102 38+3
2981 + 1203 481+90 478+255
1216+500*
49+ 19 21+11 5+4
44+8
