- Email: [email protected]
Circulating dendritic cells following burn
burns 35 (2009) 513–518
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns
Circulating dendritic cells following burn N. D’Arpa *, A. Accardo-Palumbo, G. Amato, L. D’Amelio, D. Pileri, V. Cataldo, R. Mogavero, C. Lombardo, B. Napoli, F. Conte Plastic Surgery and Burns Therapy Operating Unit, ARNAS, Civic Hospital, Piazza Leotta 4, 90127 Palermo, Italy
article info
abstract
Article history:
Burns are associated with immune suppression and subsequent development of sepsis.
Accepted 29 May 2008
Dendritic cells (DCs) are potent antigen-presenting cells that serve as a critical link between the innate and acquired immune systems, and are essential in coordinating the host
Keywords:
response to pathogens. Using multicolour flow cytometry, the percentages of LIN +
+
DR+
+
Dendritic cells
CD11c myeloid (mDC) and LIN DR CD123 plasmacytoid (pDC) subsets were determined
Sepsis
in peripheral blood from 32 people (15 septic and 5 non-septic burn victims and 12 age- and
Burn
gender-matched healthy controls, up to 20 days from injury). Analysis revealed significant
Myeloid cells
reductions in circulating mDCs and pDCs in survivor as well as non-survivor septic cases
Plasmacytoid cells
compared with non-septic cases and controls ( p < 0.001). These findings suggest that deficiencies in mDCs and pDC subsets are related to sepsis following severe burn, and may contribute to immunosuppression among burn victims. Published by Elsevier Ltd and ISBI.
1.
Introduction
Sepsis represents an important cause of mortality among people with severe burns, and correlates with age and with extent of burn [1]. Immune dysfunction has been implicated as contributing to the development of the sepsis after thermal injury [2]. Defects in innate and acquired immunity have been reported among burn victims, including depression of Th1 and increased Th2 cytokine production, macrophage dysfunction, altered NK and T-cell activities and depressed cytotoxic response [3–6]. Dendritic cells (DCs) are potent antigen-presenting cells (APCs), serve as a critical link between the innate and acquired immune systems and are essential in coordinating the host response to microorganisms. Immature DCs capture and process the antigen in inflammatory tissues and subsequently present the processed antigen to naive T cells in lymphoid tissues, to generate effector T cells [7]. Blood DCs have been divided into myeloid (mDC) and plasmacytoid (pDC) subsets, on the basis of differences in phenotype markers and function. * Corresponding author. Tel.: +39 091 666 3600; fax: +39 091 666 3708. E-mail address: [email protected] (N. D’Arpa). 0305-4179/$36.00 . Published by Elsevier Ltd and ISBI. doi:10.1016/j.burns.2008.05.027
In particular, the CD11c DCs, which express high levels of CD123, are designated plasmacytoid (pDCs), whereas the CD11c+ CD123 subsets are described as myeloid (mDCs). The mDCs have greater T-cell stimulator activity, whereas the pDCs produce elevated amounts of IFN-g [8]. DCs have been shown capable of rapid deletion during infection, and this process could be accelerated in sepsis. In vivo murine and human studies increasingly underline the importance of the loss of DCs during sepsis [9,10]. However, little is known about the percentages of circulating DCs and their subsets in burn cases. Our preliminary data demonstrated that percentages of DCs were reduced in peripheral blood from septic compared with non-septic burn victims [11]. As DCs are the major initiators of specific immune responses to pathogens, we hypothesised that an alteration in DC percentages might contribute to immune suppression following burn. In this study, we investigated correlations between the presence of sepsis and reduction of specific DC subsets after severe burn.
514
burns 35 (2009) 513–518
2.
Materials and methods
2.1.
Participants
Venous peripheral blood (5 ml) from 20 burn victims (17 men and 3 women; mean total body surface area burned 15–80%; mean age 50.2 14.6 years) and from 12 healthy people (5 men, 7 women; mean age 43 7.5 years) was collected into heparinised tubes. Samples from the burned group were collected at 1, 3, 7, 10, 14 and 20 days after burn and processed by flow cytometry to study percentages of DCs and their subsets. Burn victims were not treated with antibiotics until sepsis occurred; 15 patients developed sepsis and 4 of these died after a mean 15 13 days. Informed consent for DC analysis was obtained from all participants.
2.2.
Antibodies
The monoclonal antibodies (MoAbs) used for staining DCs were combined to prepare a lineage cocktail with FITClabelled anti-CD3, anti-CD14, anti-CD19, anti-CD20 and antiCD56 MoAbs with PerCP-labelled anti-HLA-DR MoAb (all from Becton Dickinson, Mountain View, CA, USA). In addition, PElabelled anti-CD11c and PE-labelled anti-CD123, with PElabelled IgG isotype control MoAbs (Becton Dickinson, Mountain View, CA, USA), were used to identify mDC and pDC subsets, respectively [12].
2.3.
Flow cytometry analysis
The fresh samples of venous heparinised blood were incubated with MoAbs for 30 min on ice and washed twice in phosphate-buffered saline (PBS), containing 0.1% (w/v) BSA and 0.1% (w/v) NaN3. After staining, the cells were fixed with 1% (w/v) paraformaldehyde in PBS for 30 min at room temperature before flow cytometry analysis. A cocktail of antibodies was used to identify LIN cells, which lack CD3, CD19, CD14, CD20 and CD56 markers and which are DR+. Within the LIN DR+ cells is the DC population. To identify CD11C+ and CD123+ subsets among LIN DR+ DC cells, the gate was set on LIN DR+ cells and the percentages of CD11c+PE and CD123+ PE cells were estimated using separate tubes. Three-colour flow cytometry analysis was performed using a FACScan (Becton Dickinson, Mountain View, CA, USA). At least 100,000 cells (events) were acquired for each sample. DCs were expressed as percentages of cells within the LIN DR+ gate. The acquired data were analysed using the CellQuest software program (Becton Dickinson, Mountain View, CA, USA).
2.4.
Other laboratory variables
Leukocyte and lymphocyte counts were determined by standard procedures using an automated haematology analyser (Sysmex XT 1800 L; www.mdheme.com).
Student’s t-test. Differences were considered significant when p < 0.05.
3.
Results
3.1.
Participant characteristics
Characteristics of participants are summarised in Table 1. Whereas the development of sepsis was correlated to the extent of the burned area, age was a determinant factor distinguishing survivors from non-survivors among burn victims, who were classified as septic if one of the following criteria was present: blood or tissue culture containing bacteria and fungi; hyperthermia (>38 8C); altered mental status; haemodynamic instability usually requiring vasopressors.
Signs of sepsis were observed among 15 of 20 burn victims after a mean 3 1 days. The mean age of non-survivor septic patients was higher rather than that of survivor septic patients ( p < 0.05), and the mean percentage of total body surface area (TBSA) burned was higher among septic than among nonseptic patients ( p < 0.05).
3.2.
Initially, the staining intensity of cells within the compound gate (R1) was evaluated for LIN markers (FL1) and DR expression (FL3). Within an extended lymphocyte–monocyte light scatter gate, granulocytes as well as necrotic cells were excluded. The LIN DR+ DC population (R2) was gated (Fig. 1) and used to discriminate the CD11c+from the CD123+ DC subsets.
3.3.
Statistical analysis
Data were reported as means standard deviation (S.D.). Differences were analysed using unpaired or paired two-tailed
Percentages of LINSDR+ DC
The proportion of LIN DR+ DCs was estimated as the percentage of cells in the R2 gate (Fig. 1). We studied the percentages of DCs at admission (day 1) and over 20 days after burn. To analyse the correlation between the presence of
Table 1 – Clinical characteristics of participants. Group Survivor septic patients Non-survivor septic patients Non-septic patients Controls *
2.5.
Gating strategy
Number of participants
Mean age (years)
%TBSA burned
11
42.6 9.6*
41.7 9*
4
68 12**
46.7 25*
5 12
48 6.7* 43 7.5*
18.7 4.8*** –
Not statistically significant. Significant differences between survivor septic patients, and non-septic patients and controls. *** Significant difference between survivor and non-survivor septic patients. **
burns 35 (2009) 513–518
515
Fig. 1 – The gating strategy used to identify LINS DR+ cells. Cells in the lymphocyte-monocyte light scatter gate (A) were evaluated for expression of lineage markers LIN (FL-1) and DR (FL-2). The percentage of LINS DR+ DCs was detected in the top left quadrant (R2) (B).
sepsis and DC percentages, we divided the burned group into septic and non-septic subgroups. Fig. 2 shows the percentages of LIN DR+ DCs in these subgroups and among controls on the days indicated. One day after burn, levels of circulating DCs decreased in both subgroups compared with controls ( p < 0.001). These levels were lower among septic than among non-septic cases at all time points ( p < 0.001), as shown in Fig. 2. No statistically significant difference in DC percentages was observed between survivor and non-survivor septic participants.
the septic compared with the non-septic and the control groups (Figs. 4 and 5) at all times after thermal injury, with insignificant differences between survivors from non-survivors in the septic group. In contrast, mDC and pDC subsets were higher among the non-septic than among both the septic patients ( p < 0.001) and the controls ( p < 0.05) from 3 until 10 days after burn. By 14 days, the mDC and pDC percentages in the non-septic subgroup had returned to normal levels (Figs. 4 and 5).
3.5. 3.4.
Other laboratory variables
Percentages of CD11c+ and CD123+ DCs
The expression of CD11c and CD123 on LIN DR+ DCs was determined as shown in Fig. 3, to distinguish between mDC and pDC subsets in the peripheral blood of all participants, and the mDC and pDC percentages among burn victims were compared with those among controls. The kinetic of mDCs was similar to that of pDCs in all burn cases, suggesting that burn affects both these subsets (Figs. 4 and 5). At admission, significantly differing percentages of mDC and pDC populations in the blood were observed between the burned group and controls ( p < 0.001), indicating a marked DC loss early after burn; mDC and pDC percentages were lower in
Fig. 2 – Percentages of LINSDR+ DCs in the different groups on the days.
Total leukocyte and lymphocyte counts from each group are shown in Table 2. The total leukocyte count was higher and the lymphocyte percentage was lower in the septic subgroup compared with the other participants, but these differences rarely reached statistical significance.
4.
Discussion
Our results demonstrated a decrease of circulating DCs among burn victims at admission compared with healthy controls. Interestingly, we found a decrease in both mDC and pDC subsets in the septic but not in the non-septic subgroup at all time points in our investigation. A major finding of the present study was the dramatic decrease of mDC and pDC subsets in peripheral blood in the septic subgroup. This reduction occurred early (within day 1) after thermal injury in all burn cases, but persisted only in septic subgroup. Interestingly, in the non-septic subgroup we found that the percentages of circulating mDCs and pDCs increased after day 1 until day 10 and then returned to baseline by day 20 after burn, indicating that in this subgroup DCs were recruited into the peripheral blood from bone marrow; we postulate that the increase or maintenance of blood DC subsets reflects an appropriate response to pathogens after thermal injury.
516
burns 35 (2009) 513–518
Fig. 3 – LINS DR+ DCs were evaluated for the expression of CD123 and CD11c (R3) to identify the DC subsets. Representative examples obtained from a non-septic (A and B) and a septic burn victim (C and D) are shown.
Fig. 4 – Percentages of CD11c cells among LINSDR+ DCs in all groups on the days indicated.
Fig. 5 – Percentages of CD123 cells among LINSDR+ DCs in all groups on the days indicated.
DCs are considered vital for communication between the innate and acquired immune systems. In fact, DCs migrate throughout the body and act as sentinels by constantly sampling their environment and interacting with lymphocyte and NK cells [7,13]. The mDCs are highly efficient at priming naive T cells predominantly towards a Th1 profile, and the pDCs are more specialised effectors in innate immunity [8]. Thus, we hypothesise that reduction of mDCs and/or pDCs in peripheral blood significantly affects the immune status of
burn victims, impairing the ability of the host to eradicate microorganisms. We found that plasma levels of mDCs and pDCs were reduced all cases early after thermal injury, but subsets remained low until day 20 only among participants who developed sepsis. In contrast, in the non-septic subgroup these cell populations were restored and became even more abundant from day 7 until day 10 compared with controls, but by day 14 did not differ from those of controls. No statistically
517
burns 35 (2009) 513–518
Table 2a – Kinetic of mean leucocyte count (cells T 103 mlS1 plasma) at time points after burn, showing no significant differences between groups. Group Survivor septic Non-survivor septic Non-septic
1 day
3 days
7 days
10 days
14 days
13 6.5 9.8 6 13 2
11 4 9.5 3 11.4 0.8
16.2 7 11.7 6 12 2
10.6 5 6.5 4 11 4
13 4 8.9 3 9.4 2
Controls
20 days 9.1 2 6.5 2 7.1 2
71
Table 2b – Kinetic of mean lymphocyte count (% of leucocyte count) at time points after burn. Group Survivor septic Non-survivor septic Non-septic
1 day 8.3 5 12 2.3 10 4
3 days 10.4 6 6.6 1.3* 16.7 4*
Controls (%)
7 days
10 days
14 days
20 days
**
13 9 8.5 1.8 14.5 8
15.6 5 14 5.5 21.4 6
19.1 2 *** 6.5 2*** 18.4 2***
10.3 3 4 0.9** 16 3** 25 1.8
*
Significant difference between non-survivor septic versus non-septic groups. Significant differences between all groups. *** Significant differences between survivor versus non-survivor septic patients and between non-septic versus non-survivor septic patients. **
significant difference was observed in percentages of DCs and their subsets between survivor and non-survivor septic burn victims, indicating that DC loss was related only to the presence of sepsis and not to survival or non-survival. Furthermore, we observed that the kinetic of mDCs was similar to that of pDCs among all participants, suggesting that burn affects both these DC subsets. Sepsis following thermal injury remains a poorly understood clinical problem [1]. Burned people with sepsis are immunosuppressed, as evidenced by their development of anergy, their frequent inability to eradicate their primary infection and their propensity to acquire secondary nosocomial infections [14,15], and immune dysfunction has been implicated as a causative factor in the development of sepsis after thermal injury [2]. A multitude of significant immunological alterations, including depressed NK cell activity, decreased T-cell proliferation, altered T lymphocyte activities and cytotoxic responses, have been reported to occur both among burned people and in animal models of burn [3–6]. Transforming growth factor-beta (TGF-b), a cytokine with anti-inflammatory properties, may contribute to immunosuppression after burn. Recently Choileain showed that regulatory T cells (Treg) play a central role in suppressing Tcell responses after thermal injury and that Treg activity is mediated in part by TGF-b1 [16]. However, other mechanisms may induce immunosuppression and we suggest that DCs may have a key role. DCs are clearly important in immune activation, but little is known about their role in sepsis-induced immunosuppression [17]. In vivo murine and human studies increasingly point to the importance of the loss of DCs during sepsis [10], and previous studies have illustrated a specific decrease in the splenic DC population in septic burn cases compared with other trauma cases [9]. Equally, little is known about the percentages and characterisation of circulating DC subsets among burned people. The use of a combination of MoAbs, the so-called ‘lineage cocktail’, with an anti-DR antibody is currently used to identify LIN DR+ DCs by flow cytometry in
the peripheral circulation of humans [12]. Under these conditions, most cells of all lineages (T, B lymphocyte, NK and monocytes) can be gated out and only LIN DR+ cells (DC) be retained in the gate. Using this strategy, changes in the leukocytes cannot affect the relative numbers of dendritic cells, as described by other authors [12]. A meaningful analysis of DC cells and their subsets can be reliably performed without any previous ex vivo manipulations, which could alter the proportions and the activation state of this very small cell population. We also determined leukocyte and lymphocyte counts in peripheral blood from burn victims and healthy controls. The leucocyte kinetic showed that at no time point in our study were there any significant differences between the groups. However, results for the lymphocyte kinetic obtained at days 3, 7 and 20 after burn revealed a significant difference between the septic subgroup compared with the non-septic subgroup and controls, underlining the immunosuppression of these septic burn victims. Although the data in this study do not address the mechanisms inducing the decrease in plasma mDCs and pDCs among burned people with sepsis, various hypotheses may be proposed. As circulating levels of the DC subsets are determined by a balance between their bone marrow production, migration to peripheral or lymphoid tissues and apoptosis, it is possible that depressed bone marrow release of DC precursors could significantly affect circulating mDC and pDC levels. It has been reported in a murine burn and sepsis model that there is significant decline in DC precursors after injury [18]. Human studies have demonstrated a similar phenomenon, with peripheral blood monocytes having a decreased capacity to differentiate into immature DCs in trauma cases [19]. Decreased percentages of DC subsets in the circulation of burned people with sepsis could also be the consequence of apoptosis. It has been demonstrated that DC depletion in murine lymph nodes was due, at least, in part, to apoptosis [10]. In addition, increased migration to lymphoid tissues or to
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burns 35 (2009) 513–518
peripheral inflammatory sites might contribute to dramatic reduction of circulating DC subsets. Additional studies that address the mechanism of reduction of circulating DC subsets in septic burn cases are needed. In conclusion, burned people with sepsis exhibited a reduction in both circulating mDCs and pDCs at all time points in our study. As DCs are essential for initiation and regulation of innate and adaptive immunity, we suggest that these results could yield useful information on development of sepsis after thermal injury. Moreover, the monitoring of blood DC counts might provide an early and valuable assessment of the host response to infection and thus assist in the management of burn victims.
Conflict of interest
[7]
[8]
[9]
[10]
[11]
None. [12]
references
[1] Barrow RE, Przkora R, Hawkins HK, Barrow LN, Jeschke MG, Herndon DN. Mortality related to gender, age, sepsis, and ethnicity in severely burned children. Shock 2005;23:485–7. [2] Benjamim CF, Lundy SK, Lukacs NW, Hogaboam CM, Kunkel SL. Reversal of long-term sepsis-induced immunosuppression by dendritic cells. Blood 2005;105:3588–95. [3] Schwacha MG. Macrophages and post-burn immune dysfunction. Burns 2003;29:1–14. [4] Singh H, Abdullah A, Herndon DN. Effects of rat interleukin-2 and rat interferon gamma on the natural killer cell activity of rat spleen cells after injury. J Burn Care Rehabil 1992;13:617–22. [5] Hunt JP, Hunter CT, Brownstein MR, Giannopoulos A, Hultman CS, deSerres S, et al. The effector component of the cytotoxic T-lymphocyte response has a biphasic pattern after burn injury. J Surg Res 1998;80:243–51. [6] O’Sullivan ST, Lederer JA, Horgan AF, Chin DH, Mannick JA, Rodrick ML. Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished
[13] [14] [15]
[16]
[17] [18]
[19]
interleukin-12 production associated with decreased resistance to infection. Ann Surg 1995;222:482–92. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000;18:767–811. Pichyangkul S, Endy TP, Kalayanarooj S, Nisalak A, Yongvanitchit K, Green S, et al. A blunted blood plasmacytoid dendritic cell response to an acute systemic viral infection is associated with increased disease severity. J Immunol 2003;171:5571–8. Hotchkiss RS, Tinsley KW, Swanson PE, Grayson MH, Osborne DF, Wagner TH, et al. Depletion of dendritic cells, but not macrophages, in patients with sepsis. J Immunol 2002;168:2493–500. Efron PA, Martins A, Minnich D, Tinsley K, Ungaro R, Bahjat FR, et al. Characterization of the systemic loss of dendritic cells in murine lymph nodes during polymicrobial sepsis. J Immunol 2004;173:3035–43. D’Arpa N, Accardo-Palumbo A, Amato G, D’Amelio L, Napoli B, Pileri D, et al. Decrease of circulating dendritic cells in burn patients. Ann Burns Fire Dis 2007;20:199–202. Hoffmann TK, Mu¨ller-Berghaus J, Ferris RL, Johnson JT, Storkus WJ, Whiteside TL. Alterations in the frequency of dendritic cell subsets in the peripheral circulation of patients with squamous cell carcinomas of the head and neck. Clin Cancer Res 2002;8:1787–93. Cooper MA, Fehniger TA, Fuchs A, Colonna M, Caligiuri MA. NK cell and DC interactions. Trends Immunol 2004;25:47–52. Lederer JA, Roderick ML, Mannick JA. The effects of injury on the adaptative immune response. Shock 1999;11:153–9. Puyana JC, Pellegrini JD, De AK, Kodys K, Silva WE, Miller CL. Both T-helper-1 and T-helper-2-type lymphokines are depressed in post-trauma anergy. J Trauma 1998;44:1037– 46. Choileain N, Mac Conmara M, Zang Y, Murphy TJ, Mannick JA. Enhanced regulatory T cell activity is an element of the host response to injury. J Immunol 2006;176:225–36. Moll H. Dendritic cells and host resistance to infection. Cell Microbiol 2003;5:493–500. Sen S, Muthu K, He LK, Daud A, Jones S, Gamelli R, et al. Thermal injury and sepsis deplete precursor dendritic cells and alter their functions. J Am Coll Surg 2003;197:S39. De AK, Laudanski K, Miller-Graziano CL. Failure of monocytes of trauma patients to convert to immature dendritic cells is related to preferential macrophagecolony-stimulating factor-driven macrophage differentiation. J Immunol 2003;170:6355–62.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns
Circulating dendritic cells following burn N. D’Arpa *, A. Accardo-Palumbo, G. Amato, L. D’Amelio, D. Pileri, V. Cataldo, R. Mogavero, C. Lombardo, B. Napoli, F. Conte Plastic Surgery and Burns Therapy Operating Unit, ARNAS, Civic Hospital, Piazza Leotta 4, 90127 Palermo, Italy
article info
abstract
Article history:
Burns are associated with immune suppression and subsequent development of sepsis.
Accepted 29 May 2008
Dendritic cells (DCs) are potent antigen-presenting cells that serve as a critical link between the innate and acquired immune systems, and are essential in coordinating the host
Keywords:
response to pathogens. Using multicolour flow cytometry, the percentages of LIN +
+
DR+
+
Dendritic cells
CD11c myeloid (mDC) and LIN DR CD123 plasmacytoid (pDC) subsets were determined
Sepsis
in peripheral blood from 32 people (15 septic and 5 non-septic burn victims and 12 age- and
Burn
gender-matched healthy controls, up to 20 days from injury). Analysis revealed significant
Myeloid cells
reductions in circulating mDCs and pDCs in survivor as well as non-survivor septic cases
Plasmacytoid cells
compared with non-septic cases and controls ( p < 0.001). These findings suggest that deficiencies in mDCs and pDC subsets are related to sepsis following severe burn, and may contribute to immunosuppression among burn victims. Published by Elsevier Ltd and ISBI.
1.
Introduction
Sepsis represents an important cause of mortality among people with severe burns, and correlates with age and with extent of burn [1]. Immune dysfunction has been implicated as contributing to the development of the sepsis after thermal injury [2]. Defects in innate and acquired immunity have been reported among burn victims, including depression of Th1 and increased Th2 cytokine production, macrophage dysfunction, altered NK and T-cell activities and depressed cytotoxic response [3–6]. Dendritic cells (DCs) are potent antigen-presenting cells (APCs), serve as a critical link between the innate and acquired immune systems and are essential in coordinating the host response to microorganisms. Immature DCs capture and process the antigen in inflammatory tissues and subsequently present the processed antigen to naive T cells in lymphoid tissues, to generate effector T cells [7]. Blood DCs have been divided into myeloid (mDC) and plasmacytoid (pDC) subsets, on the basis of differences in phenotype markers and function. * Corresponding author. Tel.: +39 091 666 3600; fax: +39 091 666 3708. E-mail address: [email protected] (N. D’Arpa). 0305-4179/$36.00 . Published by Elsevier Ltd and ISBI. doi:10.1016/j.burns.2008.05.027
In particular, the CD11c DCs, which express high levels of CD123, are designated plasmacytoid (pDCs), whereas the CD11c+ CD123 subsets are described as myeloid (mDCs). The mDCs have greater T-cell stimulator activity, whereas the pDCs produce elevated amounts of IFN-g [8]. DCs have been shown capable of rapid deletion during infection, and this process could be accelerated in sepsis. In vivo murine and human studies increasingly underline the importance of the loss of DCs during sepsis [9,10]. However, little is known about the percentages of circulating DCs and their subsets in burn cases. Our preliminary data demonstrated that percentages of DCs were reduced in peripheral blood from septic compared with non-septic burn victims [11]. As DCs are the major initiators of specific immune responses to pathogens, we hypothesised that an alteration in DC percentages might contribute to immune suppression following burn. In this study, we investigated correlations between the presence of sepsis and reduction of specific DC subsets after severe burn.
514
burns 35 (2009) 513–518
2.
Materials and methods
2.1.
Participants
Venous peripheral blood (5 ml) from 20 burn victims (17 men and 3 women; mean total body surface area burned 15–80%; mean age 50.2 14.6 years) and from 12 healthy people (5 men, 7 women; mean age 43 7.5 years) was collected into heparinised tubes. Samples from the burned group were collected at 1, 3, 7, 10, 14 and 20 days after burn and processed by flow cytometry to study percentages of DCs and their subsets. Burn victims were not treated with antibiotics until sepsis occurred; 15 patients developed sepsis and 4 of these died after a mean 15 13 days. Informed consent for DC analysis was obtained from all participants.
2.2.
Antibodies
The monoclonal antibodies (MoAbs) used for staining DCs were combined to prepare a lineage cocktail with FITClabelled anti-CD3, anti-CD14, anti-CD19, anti-CD20 and antiCD56 MoAbs with PerCP-labelled anti-HLA-DR MoAb (all from Becton Dickinson, Mountain View, CA, USA). In addition, PElabelled anti-CD11c and PE-labelled anti-CD123, with PElabelled IgG isotype control MoAbs (Becton Dickinson, Mountain View, CA, USA), were used to identify mDC and pDC subsets, respectively [12].
2.3.
Flow cytometry analysis
The fresh samples of venous heparinised blood were incubated with MoAbs for 30 min on ice and washed twice in phosphate-buffered saline (PBS), containing 0.1% (w/v) BSA and 0.1% (w/v) NaN3. After staining, the cells were fixed with 1% (w/v) paraformaldehyde in PBS for 30 min at room temperature before flow cytometry analysis. A cocktail of antibodies was used to identify LIN cells, which lack CD3, CD19, CD14, CD20 and CD56 markers and which are DR+. Within the LIN DR+ cells is the DC population. To identify CD11C+ and CD123+ subsets among LIN DR+ DC cells, the gate was set on LIN DR+ cells and the percentages of CD11c+PE and CD123+ PE cells were estimated using separate tubes. Three-colour flow cytometry analysis was performed using a FACScan (Becton Dickinson, Mountain View, CA, USA). At least 100,000 cells (events) were acquired for each sample. DCs were expressed as percentages of cells within the LIN DR+ gate. The acquired data were analysed using the CellQuest software program (Becton Dickinson, Mountain View, CA, USA).
2.4.
Other laboratory variables
Leukocyte and lymphocyte counts were determined by standard procedures using an automated haematology analyser (Sysmex XT 1800 L; www.mdheme.com).
Student’s t-test. Differences were considered significant when p < 0.05.
3.
Results
3.1.
Participant characteristics
Characteristics of participants are summarised in Table 1. Whereas the development of sepsis was correlated to the extent of the burned area, age was a determinant factor distinguishing survivors from non-survivors among burn victims, who were classified as septic if one of the following criteria was present: blood or tissue culture containing bacteria and fungi; hyperthermia (>38 8C); altered mental status; haemodynamic instability usually requiring vasopressors.
Signs of sepsis were observed among 15 of 20 burn victims after a mean 3 1 days. The mean age of non-survivor septic patients was higher rather than that of survivor septic patients ( p < 0.05), and the mean percentage of total body surface area (TBSA) burned was higher among septic than among nonseptic patients ( p < 0.05).
3.2.
Initially, the staining intensity of cells within the compound gate (R1) was evaluated for LIN markers (FL1) and DR expression (FL3). Within an extended lymphocyte–monocyte light scatter gate, granulocytes as well as necrotic cells were excluded. The LIN DR+ DC population (R2) was gated (Fig. 1) and used to discriminate the CD11c+from the CD123+ DC subsets.
3.3.
Statistical analysis
Data were reported as means standard deviation (S.D.). Differences were analysed using unpaired or paired two-tailed
Percentages of LINSDR+ DC
The proportion of LIN DR+ DCs was estimated as the percentage of cells in the R2 gate (Fig. 1). We studied the percentages of DCs at admission (day 1) and over 20 days after burn. To analyse the correlation between the presence of
Table 1 – Clinical characteristics of participants. Group Survivor septic patients Non-survivor septic patients Non-septic patients Controls *
2.5.
Gating strategy
Number of participants
Mean age (years)
%TBSA burned
11
42.6 9.6*
41.7 9*
4
68 12**
46.7 25*
5 12
48 6.7* 43 7.5*
18.7 4.8*** –
Not statistically significant. Significant differences between survivor septic patients, and non-septic patients and controls. *** Significant difference between survivor and non-survivor septic patients. **
burns 35 (2009) 513–518
515
Fig. 1 – The gating strategy used to identify LINS DR+ cells. Cells in the lymphocyte-monocyte light scatter gate (A) were evaluated for expression of lineage markers LIN (FL-1) and DR (FL-2). The percentage of LINS DR+ DCs was detected in the top left quadrant (R2) (B).
sepsis and DC percentages, we divided the burned group into septic and non-septic subgroups. Fig. 2 shows the percentages of LIN DR+ DCs in these subgroups and among controls on the days indicated. One day after burn, levels of circulating DCs decreased in both subgroups compared with controls ( p < 0.001). These levels were lower among septic than among non-septic cases at all time points ( p < 0.001), as shown in Fig. 2. No statistically significant difference in DC percentages was observed between survivor and non-survivor septic participants.
the septic compared with the non-septic and the control groups (Figs. 4 and 5) at all times after thermal injury, with insignificant differences between survivors from non-survivors in the septic group. In contrast, mDC and pDC subsets were higher among the non-septic than among both the septic patients ( p < 0.001) and the controls ( p < 0.05) from 3 until 10 days after burn. By 14 days, the mDC and pDC percentages in the non-septic subgroup had returned to normal levels (Figs. 4 and 5).
3.5. 3.4.
Other laboratory variables
Percentages of CD11c+ and CD123+ DCs
The expression of CD11c and CD123 on LIN DR+ DCs was determined as shown in Fig. 3, to distinguish between mDC and pDC subsets in the peripheral blood of all participants, and the mDC and pDC percentages among burn victims were compared with those among controls. The kinetic of mDCs was similar to that of pDCs in all burn cases, suggesting that burn affects both these subsets (Figs. 4 and 5). At admission, significantly differing percentages of mDC and pDC populations in the blood were observed between the burned group and controls ( p < 0.001), indicating a marked DC loss early after burn; mDC and pDC percentages were lower in
Fig. 2 – Percentages of LINSDR+ DCs in the different groups on the days.
Total leukocyte and lymphocyte counts from each group are shown in Table 2. The total leukocyte count was higher and the lymphocyte percentage was lower in the septic subgroup compared with the other participants, but these differences rarely reached statistical significance.
4.
Discussion
Our results demonstrated a decrease of circulating DCs among burn victims at admission compared with healthy controls. Interestingly, we found a decrease in both mDC and pDC subsets in the septic but not in the non-septic subgroup at all time points in our investigation. A major finding of the present study was the dramatic decrease of mDC and pDC subsets in peripheral blood in the septic subgroup. This reduction occurred early (within day 1) after thermal injury in all burn cases, but persisted only in septic subgroup. Interestingly, in the non-septic subgroup we found that the percentages of circulating mDCs and pDCs increased after day 1 until day 10 and then returned to baseline by day 20 after burn, indicating that in this subgroup DCs were recruited into the peripheral blood from bone marrow; we postulate that the increase or maintenance of blood DC subsets reflects an appropriate response to pathogens after thermal injury.
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Fig. 3 – LINS DR+ DCs were evaluated for the expression of CD123 and CD11c (R3) to identify the DC subsets. Representative examples obtained from a non-septic (A and B) and a septic burn victim (C and D) are shown.
Fig. 4 – Percentages of CD11c cells among LINSDR+ DCs in all groups on the days indicated.
Fig. 5 – Percentages of CD123 cells among LINSDR+ DCs in all groups on the days indicated.
DCs are considered vital for communication between the innate and acquired immune systems. In fact, DCs migrate throughout the body and act as sentinels by constantly sampling their environment and interacting with lymphocyte and NK cells [7,13]. The mDCs are highly efficient at priming naive T cells predominantly towards a Th1 profile, and the pDCs are more specialised effectors in innate immunity [8]. Thus, we hypothesise that reduction of mDCs and/or pDCs in peripheral blood significantly affects the immune status of
burn victims, impairing the ability of the host to eradicate microorganisms. We found that plasma levels of mDCs and pDCs were reduced all cases early after thermal injury, but subsets remained low until day 20 only among participants who developed sepsis. In contrast, in the non-septic subgroup these cell populations were restored and became even more abundant from day 7 until day 10 compared with controls, but by day 14 did not differ from those of controls. No statistically
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Table 2a – Kinetic of mean leucocyte count (cells T 103 mlS1 plasma) at time points after burn, showing no significant differences between groups. Group Survivor septic Non-survivor septic Non-septic
1 day
3 days
7 days
10 days
14 days
13 6.5 9.8 6 13 2
11 4 9.5 3 11.4 0.8
16.2 7 11.7 6 12 2
10.6 5 6.5 4 11 4
13 4 8.9 3 9.4 2
Controls
20 days 9.1 2 6.5 2 7.1 2
71
Table 2b – Kinetic of mean lymphocyte count (% of leucocyte count) at time points after burn. Group Survivor septic Non-survivor septic Non-septic
1 day 8.3 5 12 2.3 10 4
3 days 10.4 6 6.6 1.3* 16.7 4*
Controls (%)
7 days
10 days
14 days
20 days
**
13 9 8.5 1.8 14.5 8
15.6 5 14 5.5 21.4 6
19.1 2 *** 6.5 2*** 18.4 2***
10.3 3 4 0.9** 16 3** 25 1.8
*
Significant difference between non-survivor septic versus non-septic groups. Significant differences between all groups. *** Significant differences between survivor versus non-survivor septic patients and between non-septic versus non-survivor septic patients. **
significant difference was observed in percentages of DCs and their subsets between survivor and non-survivor septic burn victims, indicating that DC loss was related only to the presence of sepsis and not to survival or non-survival. Furthermore, we observed that the kinetic of mDCs was similar to that of pDCs among all participants, suggesting that burn affects both these DC subsets. Sepsis following thermal injury remains a poorly understood clinical problem [1]. Burned people with sepsis are immunosuppressed, as evidenced by their development of anergy, their frequent inability to eradicate their primary infection and their propensity to acquire secondary nosocomial infections [14,15], and immune dysfunction has been implicated as a causative factor in the development of sepsis after thermal injury [2]. A multitude of significant immunological alterations, including depressed NK cell activity, decreased T-cell proliferation, altered T lymphocyte activities and cytotoxic responses, have been reported to occur both among burned people and in animal models of burn [3–6]. Transforming growth factor-beta (TGF-b), a cytokine with anti-inflammatory properties, may contribute to immunosuppression after burn. Recently Choileain showed that regulatory T cells (Treg) play a central role in suppressing Tcell responses after thermal injury and that Treg activity is mediated in part by TGF-b1 [16]. However, other mechanisms may induce immunosuppression and we suggest that DCs may have a key role. DCs are clearly important in immune activation, but little is known about their role in sepsis-induced immunosuppression [17]. In vivo murine and human studies increasingly point to the importance of the loss of DCs during sepsis [10], and previous studies have illustrated a specific decrease in the splenic DC population in septic burn cases compared with other trauma cases [9]. Equally, little is known about the percentages and characterisation of circulating DC subsets among burned people. The use of a combination of MoAbs, the so-called ‘lineage cocktail’, with an anti-DR antibody is currently used to identify LIN DR+ DCs by flow cytometry in
the peripheral circulation of humans [12]. Under these conditions, most cells of all lineages (T, B lymphocyte, NK and monocytes) can be gated out and only LIN DR+ cells (DC) be retained in the gate. Using this strategy, changes in the leukocytes cannot affect the relative numbers of dendritic cells, as described by other authors [12]. A meaningful analysis of DC cells and their subsets can be reliably performed without any previous ex vivo manipulations, which could alter the proportions and the activation state of this very small cell population. We also determined leukocyte and lymphocyte counts in peripheral blood from burn victims and healthy controls. The leucocyte kinetic showed that at no time point in our study were there any significant differences between the groups. However, results for the lymphocyte kinetic obtained at days 3, 7 and 20 after burn revealed a significant difference between the septic subgroup compared with the non-septic subgroup and controls, underlining the immunosuppression of these septic burn victims. Although the data in this study do not address the mechanisms inducing the decrease in plasma mDCs and pDCs among burned people with sepsis, various hypotheses may be proposed. As circulating levels of the DC subsets are determined by a balance between their bone marrow production, migration to peripheral or lymphoid tissues and apoptosis, it is possible that depressed bone marrow release of DC precursors could significantly affect circulating mDC and pDC levels. It has been reported in a murine burn and sepsis model that there is significant decline in DC precursors after injury [18]. Human studies have demonstrated a similar phenomenon, with peripheral blood monocytes having a decreased capacity to differentiate into immature DCs in trauma cases [19]. Decreased percentages of DC subsets in the circulation of burned people with sepsis could also be the consequence of apoptosis. It has been demonstrated that DC depletion in murine lymph nodes was due, at least, in part, to apoptosis [10]. In addition, increased migration to lymphoid tissues or to
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peripheral inflammatory sites might contribute to dramatic reduction of circulating DC subsets. Additional studies that address the mechanism of reduction of circulating DC subsets in septic burn cases are needed. In conclusion, burned people with sepsis exhibited a reduction in both circulating mDCs and pDCs at all time points in our study. As DCs are essential for initiation and regulation of innate and adaptive immunity, we suggest that these results could yield useful information on development of sepsis after thermal injury. Moreover, the monitoring of blood DC counts might provide an early and valuable assessment of the host response to infection and thus assist in the management of burn victims.
Conflict of interest
[7]
[8]
[9]
[10]
[11]
None. [12]
references
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