J. Y. Lu, Department of Pathology, the First Affiliated Hospital of General Hospital of PLA, 51 Fucheng Road, Beijing 100048, China. E-mail: firstname.lastname@example.org
Depletion and dysfunction of dendritic cells in the lung can induce local immunoparalysis, which often leads to multiple organ dysfunction syndrome (MODS)-associated mortality. A therapeutic strategy that reverses this immunoparalysis is required. In the present study, we examined the effects of in vivo Fms-like tyrosine kinase 3 ligand (Flt3L) treatment on zymosan (zym)-induced secondary lung injury and dendritic cell (DC) immunoparalysis. BALBc mice were divided randomly into four groups (20/group): (1) sham [intraperitoneal (i.p.) saline] + vehicle [subcutaneous (s.c.) 0·01% mouse serum albumin]; (2) sham + Flt3L (s.c.); (3) zym (i.p.) + vehicle; and (4) zym + Flt3L. Injections were for 9 consecutive days; 12 days later we examined: survival rate (monitored for 12 days); lung tissue histopathology (haematoxylin and eosin staining); plasma indices of lung function (pH, PaO2, PaCO2, HCO3–); DC subsets in lung tissue; and lung DCs production of interleukin (IL)-12p70 and IL-10. Zym administration resulted in increased mortality associated with significant lung histopathological changes and abnormal blood gas indices; however, these pathological changes were ameliorated by Flt3L treatment. Zym injections also resulted in significant reductions in DC subsets recovered from lungs [CD11c+major histocompatibility complex (MHC)-II/I-Ad+, CD11c+CD11b+ and CD11c+B220+]. Importantly, in-vivo Flt3L treatment reversed these trends for DC immunoparalysis by increasing the percentages of recovered DC subsets concomitant with increased DC production of IL-12 p70 and decreased IL-10 production. These results suggest that Flt3L may have therapeutic potential for reversing DC immunoparalysis and ameliorating lung injury secondary to MODS.
Multiple organ dysfunction syndrome (MODS), also known as multiple organ failure, is the progressive functional deterioration in several organ systems resulting from sepsis, septic shock, multiple trauma, severe burns or systemic inflammatory response syndrome (SIRS). MODS often begins with lung failure, followed by failures of the gut, kidneys and liver. Despite major advances in critical care medicine, MODS remains a serious health problem with dramatically high mortality rates .
Inflammation was thought initially to play a primary role in the body's response to MODS . Consequently, numerous therapies were based on the concept that anti-inflammatory agents should mitigate effectively against a morbid state, although numerous clinical trial failures suggested that the pathological mechanisms underlying MODS were not completely understood . However, recent studies have shown that MODS is also the result of a state of immunosuppression, which appears to contribute significantly to the poor outcomes among these critically ill individuals [4–6]. In particular, the respiratory system is most susceptible to secondary infections after an overwhelming SIRS, and pulmonary complications often lead to significant mortality associated with MODS [7–9]. Given the extraordinarily high mortality rates with MODS , a therapeutic strategy that reverses pulmonary immunosuppression is required.
Dendritic cells (DCs), antigen-presenting cells, are a critical, integrative link between the innate and adaptive immune systems [11,12]. Recent studies have suggested that DCs are not only affected (killed) during SIRS, but also contribute to the development of immune suppression or immunoparalysis in sepsis or septic shock, which is probably a key factor contributing to MODS morbidity and mortality [13–16]. The most prominent changes in DCs during sepsis are their extensive depletion and the sustained functional impairment of these cells [17–19]. Agents that can increase the numbers of DCs and improve their functional status might be useful therapeutics against organ injury.
Fms-like tyrosine kinase 3 ligand (Flt3L) is a haematopoietic growth factor whose receptor, CD135, is a member of the type III receptor tyrosine kinase family . Previous studies have shown that Flt3L administered to mice could alter the proportions and functions of DC subsets in the lungs and spleens [21–25]. However, the potential of Flt3L as a therapeutic agent against organ injury and immunological dysfunction in MODS has not been examined.
Thus, we investigated whether in-vivo treatment with Flt3L could reverse the immunoparalysis of lung DCs and reduce the development of secondary lung injury in a zymosan-induced generalized inflammation (ZIGI) model. Zymosan is a non-bacterial, non-endotoxic agent from yeast cell wall extracts. When injected intraperitoneally (i.p.), it causes acute peritonitis and organ injury in experimental animals and mimics both the sequence and the processes of organ injury in human MODS of non-septic origin . Specifically, we examined any effects of Flt3L treatment for zymosan-induced: (1) mortality; (2) body weight loss; (3) changes in the numbers and function of pulmonary DC subsets; (4) plasma biochemical indices of pulmonary function; and (5) histopathological changes in the lung. Our results suggest that in-vivo treatment with Flt3L can reverse lung DC immunoparalysis and ameliorate zymosan-induced secondary lung injury.
Materials and methods
Adult male BALB/c (I-Ad MHC class II) mice (6–8 weeks old; 22–25 g) were purchased from the Laboratory Animal Centre, Beijing, China. The animals were allowed to acclimatize for at least 7 days before use in experiments. They were housed in a controlled environment and provided standard rodent chow and water, but were fasted overnight prior to experiments. Animal care complied with Chinese regulations (SYXK2002-006) for the protection of animals used for experimental and other scientific purposes.
Mice were divided randomly into four groups (20/ group), as follows: (1) sham+vehicle: mice were injected subcutaneously (s.c.) with a vehicle [0·01% mouse serum albumin (MSA); Sigma-Aldrich, St Louis, MO, USA] used for recombinant human Flt3L (Pepro Tech, Rocky Hill, NJ, USA) and injected i.p. with saline solution (sham treatment); (2) zym+vehicle: mice were injected i.p. with zymosan (zym) suspended in saline solution (900 mg/kg; Sigma-Aldrich) and with vehicle (0·01% MSA) used for Flt3L; (3) sham+Flt3L: mice received subcutaneous injections of Flt3L (10 µg in 0·01% MSA) once daily for 9 consecutive days at 24 h after saline administration; (4) zym+Flt3L: mice received intraperitoneal zymosan (900 mg/kg, suspended in saline solution) and subcutaneous Flt3L (10 µg in 0·01% MSA) for 9 consecutive days at 24 h after zymosan administration. These mice were used to quantify lung function and injury, and to assess lung DC subsets and functions at 12 days after administering zymosan (details given below).
In another set of experiments, mice (20/group) were divided randomly into the same four groups as above and monitored for body weight loss and mortality for 12 days after zymosan or saline administration [26,33].
Quantification of lung function and injury
Blood samples were obtained 12 days after zymosan or saline injection and analysed immediately using standard laboratory techniques. To evaluate the acid base balance and blood gases (indicators of lung injury), arterial blood levels of pH, PaO2, PaCO2 and bicarbonate ion (HCO3–) were determined using a pH/blood gas analyser, as described previously .
Histopathological evaluation of lung tissue
Lungs were harvested 12 days after zymosan or saline injection. Whole-lung samples for histological examination were excised, perfused with 10% formalin and placed into fresh formalin for an additional 24 h. Routine histological techniques were used for paraffin-embedded tissue. Lung sections (7 µm) were stained with haematoxylin and eosin (H&E). Images were captured with an Olympus BX40F microscope (Olympus, Melville, NY, USA) and digital photographs were obtained with a Sony 3CCD colour video camera (Sony, Tokyo, Japan). The remaining lung sections were stained with anti-mouse CD205 using a standard immunohistochemistry protocol [horseradish peroxidase (HRP) – peroxide – diaminobenzidine (DAB)].
The sections for immunostaining were read by senior pathologists. The counting and analysis for CD205 immunohistochemical staining involved the following. Five sections per mouse per treatment group were selected to count cells with CD205-positive staining. Each section had cell counts taken for five visual fields under ×400 magnification. We considered only CD205-positive cells that were located in the alveolar septum and pulmonary interstitial tissues and had typical DC morphologies with dendrite processes on their cell surfaces. The numbers of CD205-positive cells on each section after summing the CD205-positive cells in five visual fields under ×400 magnification were compared statistically among the four groups.
Preparation of low-density lung cells
Low-density lung cells were prepared as described previously [25,35]. Briefly, mice were injected i.p. with 150 U heparin and airways were lavaged with cold phosphate-buffered saline (PBS) containing 0·6 mM ethylenediamine tetraacetic acid (EDTA) using an intratracheal cannula. The pulmonary vasculature was perfused with sterile saline to remove peripheral blood cells. The lavaged, perfused lungs were minced and enzyme-treated at 37°C for 90 min in RPMI-1640 (HyClone Laboratories, Logan, UT, USA) containing collagenase type I (330 U/ml; Worthington Biochemical, Lakewood, NJ, USA), type IV bovine pancreatic DNase I (50 U/ml; Sigma-Aldrich) and 10% fetal bovine serum (FBS) (HyClone Laboratories). Tissues were incubated at 37°C in a sterile test tube for 90 min. During incubation, tissues were pipetted vigorously every 30 min. After incubation, the digestion mixture was passed through a 250 µm nylon mesh to remove undigested tissue. Lung cells were resuspended in high-density Percoll (ρ = 1·075 g/ml), overlaid with an equal volume of lower density Percoll (ρ = 1·030 g/ml) and centrifuged at ×400 g for 20 min. Low-density lung cells, enriched for mononuclear cells, were recovered from the 1·075/1·030 Percoll interface and washed with Hanks' balanced salt solution (HBSS).
Flow cytometry analysis
Low-density lung cells were first pre-incubated with immunoglobulin constant fragment (Fc)-receptor-blocking antibody (anti-CD16/CD32) for 10 min to reduce non-specific binding. Subsequently, cells were labelled with a combination of monoclonal antibodies (mAbs) that included phycoerythrin (PE)-anti-CD11c(HL3), fluorescein isothiocyanate (FITC)-anti-CD11b(M1/70), peridinin–chlorophyll protein (PerCP)-anti-CD45R/B220(RA3-6B2) and AlexaFluor®647-anti-I-Ad(39-10-18) in Dulbecco's PBS and 0·2% bovine serum albumin (BSA) for 30 min at 4°C in the dark. Cells were washed three times with PBS containing 2% FBS and 40 µg/ml EDTA after each incubation step. The appropriate immunoglobulin (Ig)G isotypes were used as controls. All antibodies and IgG isotypes were purchased from BD PharMingen (San Diego, CA, USA). Cells were fixed in 1% paraformaldehyde and kept in the dark at 4°C. A BD Biosciences fluorescence activated cell sorter (FACS)Calibur (Mountain View, CA, USA) was used for data acquisition and FlowJo software (Tree Star Inc., Ashland, OR, USA) was used for data analysis.
Lung DC isolation and in-vitro stimulation
Low-density lung cells, isolated from the pooled lungs of each group at 12 days after zymosan or saline administration, were resuspended in RPMI-1640. Macrophages were removed from the CD11c cell population by adherence to plastic overnight at 37°C in a 5% CO2 atmosphere. Then, non-adherent cells were collected and enriched with anti-CD11c magnetic beads and positive selection MS+ columns according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA, USA). DCs purity was determined either by cell morphology using cytospin followed by haematoxylin and eosin (H&E) staining or by staining with PE-labelled CD11c followed by flow cytometry analysis. Purified DCs were counted with a haemocytometer and then diluted to 5 × 106/ml. Cell aliquots (100 µl) were added to 48-well plates in RPMI-1640 supplemented with 10% FCS. Lipopolysaccharide (LPS; 2 µg/ml) was added to these DCs. After 24 h of stimulation, cell-free supernatants were collected and stored at −80°C until used for further studies.
The amounts of IL-12p70 and IL-10 secreted by LPS-stimulated DCs in culture supernatants were determined using standardized sandwich enzyme-linked immunosorbent assay (ELISA) kits for mouse interleukin (IL)-12p70 and IL-10. Kits were from BD PharMingen and used according to the manufacturer's recommendations. The limits of sensitivity for IL-12p70 and IL-10 were 8·8 and 8·2 pg/ml, respectively.
Mortality rates among the four groups at each time-point were compared by Fisher's exact test. Bonferroni's method was used to adjust for multiple comparisons if overall mortality rate were significantly different. Results for body weights are given as means ± standard errors of the mean (s.e.m.) and were compared by one-way analysis of variance (anova) and t-test. Results for other continuous variables are given as medians (25th, 75th percentiles). A Kruskal–Wallis test was used to compare the effects of different treatments and Bonferroni's method was used to adjust for multiple comparisons. All assessments were two-sided at the 0·05 level. Statistical analyses used sas version 9·2 software (SAS Institute Inc., Cary, NC, USA).
Flt3L effects on mouse body weight loss and survival rate
As described in Materials and methods, four groups of mice (20/group) were monitored for mortality beginning 12 days after administering either zym or sham and treated with either a vehicle or with Flt3L. Zym treatment significantly reduced mouse body weights in comparison to the two sham groups (Fig. 1a). However, Flt3L treatment significantly decreased the zym-induced body weight loss. For the two sham (saline-injected) groups (either vehicle or Flt3L treatment), all mice survived to the end of the observation period (Fig. 1b). In the zym-treated groups, only 20% of the mice treated with vehicle had survived at day 12 after the treatment, whereas 50% of the mice treated with Flt3L survived (Fig. 1b).
Flt3L effects on lung injury
We also examined the effects of Flt3L on lung injury at 12 days after zym or sham administration by H&E-stained tissue sections for histological examinations (representative photomicrographs are shown in Fig. 2). No histological alterations were observed for sham mice (Fig. 2a). Flt3L treatment in sham mice did not change histological structures dramatically, but there were increased interstitial cells and alveolar spaces, which indicated that Flt3L might increase DCs in pulmonary interstitium (Fig. 2b). However, mice that received zym had significant pathological changes in their lung tissues; these sections revealed interstitial oedema with massive infiltrations of inflammatory cells, significant extravasation of red cells, and the pulmonary architecture was severely damaged (Fig. 2c). By comparison, treatment with Flt3L resulted in significant reductions in pulmonary injury and these morphological alterations (Fig. 2d).
As summarized in Fig. 3, the effects of Flt3L treatment on lung injury were also assessed by determining the following indices for blood samples obtained 12 days after treatments: (a) PaO2, (b) PaCO2, (c) HCO3– and (d) pH. In comparison to sham mice without Flt3L treatment, zym treatment significantly worsened the results for PaO2, PaCO2, HCO3 and pH (P = 0·002, P = 0·005, P = 0·002 and P = 0·002, respectively). Treatment with Flt3L reversed these zym-induced effects significantly, except for PaCO2 (PaO2: P = 0·010; PaCO2: P = 0·054; HCO3–: P = 0·019; pH: P = 0·024). No significant differences were found for all indices between sham with Flt3L treatment and sham with vehicle treatment, or between sham with vehicle treatment and zym with Flt3L treatment.
Flt3L effects on DCs expressing CD11c and I-Ad
Figure 4 summarizes the percentages of recovered lung DCs that expressed CD11c and I-Ad at 12 days after treatment. The percentages of pulmonary DCs expressing CD11c and I-Ad after Flt3L treatment were significantly higher than after vehicle treatment in both the sham and zym groups (P = 0·002 and P = 0·007, respectively). However, for mice that received vehicle treatment, sham mice had a significantly higher percentage of DCs expressing CD11c and I-Ad than did zym mice (P = 0·005). There were no significant differences in the percentages of these cells for mice that received Flt3L treatment between the sham and zym groups.
Flt3L increases CD205-positive DCs in lung alveoli
We also examined CD205 (DERC205), a kind of C type agglutinin of 205 kDa molecular weight. CD205 is a novel endocytic receptor used by DC to direct captured antigens from the extracellular space to specialized antigen-processing molecules; it mediates antigen uptake and presentation and cross-presentation to T cells.
As described in Methods, for these evaluations we considered only CD205+ cells that were located in the alveolar septum and pulmonary interstitial tissues and had typical DC morphologies with dendrite processes on their cell surfaces. There were very few CD205+ DCs in the lung tissues from mice in the sham+vehicle group, which were scattered in the alveolar interstitium (Fig. 5a). In this group, the average number of CD205+DCs per field at ×400 magnification was 14·8 ± 1·3 (average of five microscopic fields/tissue section; Fig. 5e). CD205+ DCs increased significantly in the sham+Flt3L group; these cells were distributed widely in the alveolar interstitium with an average number of CD205+DCs per field of 46·0 ± 3·4 (Fig. 5b). The average for the sham+Flt3L group was significantly higher than for the sham+vehicle group (Fig. 5e; P = 0·012).
In the zym+vehicle group, alveoli were disrupted and there were few CD205+ DCs: average/field = 17·4 ± 2·2 (Fig. 5c). However, there were more CD205+ DCs in the lungs of the zym+Flt3L mice (Fig. 5d; average/field = 33·6 ± 3·6) compared to the zym+vehicle group (Fig. 5e; P = 0·012). There was a trend for fewer CD205+ DCs in the lungs of the zym+Flt3L mice (Fig. 5d; 33·6 ± 3·6) compared to sham+Flt3L mice (Fig. 5b; 46·0 ± 3·4), although this difference was not statistically significant (Fig. 5e).
Flt3L effects on CD11c+CD11b+ DCs and CD11c+B220+ DCs
Regarding CD11c+CD11b+ DCs, as shown in Fig. 6a, Flt3L treatment resulted in significantly higher percentages of these cells than did vehicle treatment in both the sham and zym groups (P = 0·002 and P = 0·005, respectively). The percentage of these cells in mice after vehicle treatment was significantly higher in the sham than in the zym groups (P = 0·003). Flt3L treatment resulted in no significant difference between the sham and zym groups.
However, as shown in Fig. 6b, comparisons of CD11c+B220+ DCs between any two groups were significantly different: Flt3L versus vehicle in sham groups: P = 0·002; Flt3L versus vehicle in ZYM groups: P = 0·005; zym versus sham with vehicle treatment: P = 0·003; zym versus sham with Flt3L treatment: P = 0·002.
Flt3L effects on DCs secretion of IL-12p70 and IL-10 after zym treatment
The effects of Flt3L treatment on IL-12p70 and IL-10 secretion by cultured, LPS-stimulated DCs (CD11c+) after zym administration are shown in Fig. 7. Flt3L treatment resulted in higher levels of IL-12p70 produced by CD11c+ DCs than did vehicle treatment (Fig. 7a) and lower levels of IL-10 (Fig. 7b).
This study demonstrated that zymosan administration caused significant lung injury (histological changes), which resulted in lung dysfunction (biochemical indices) during the MODS stage. Compared with controls, Flt3L treatment ameliorated most of the effects induced by zymosan on lung injury and provided significant improvements in the biochemical indices of lung function, body weight loss and survival rates at the MODS stage. Importantly, Flt3L treatment resulted in increased numbers of pulmonary DCs, preferentially of the CD11c+CD11b+ myeloid and CD11c+CD45R/B220+ plasmacytoid subsets, which were functionally more active and phenotypically more mature. Flt3L treatment also amplified the repopulation of pulmonary DCs expressing MHC-II(I-Ad). In addition, Flt3L treatment increased CD205-positive DC numbers significantly in the lung, which could enhance the antigen-presenting and T cell-activating capabilities of DCs and could be used to treat DC immunosuppression. Flt3L also reversed the reductions in myeloid and plasmacytoid pulmonary DC subsets in zymosan-induced MODS. Thus, Flt3L appears to have a profound impact on reversing MODS-associated immunoparalysis and ameliorating lung injury.
Recent reports have demonstrated that systemic administration of Flt3L resulted in dramatic increases in functionally active DCs in a variety of organs, including the lung, spleen and lymph nodes [21–25]. We showed that Flt3L injections into mice increased the numbers of lung DCs, predominantly the myeloid subset, which were functionally more active and phenotypically more mature than lung DCs from vehicle-treated mice. Other studies have reported that Flt3L-induced DCs expressed higher levels of MHC-II, CD86 and CD40, produced higher levels of IL-12 and were more efficient at stimulating T cell proliferation than normal DCs from mice that did not receive Flt3L [27–29]. Our study showed that consecutive daily Flt3L injections could effectively increase the percentages of pulmonary myeloid and plasmacytoid DC subsets compared with vehicle-treated groups. Further, the numbers of DCs expressing MHC-II/I-Ad increased dramatically and produced higher levels of IL-12p70 after LPS stimulation in vitro.
We also showed that, during the MODS stage of the ZIGI model, mice had lower numbers of two DC subpopulations in the lungs: CD11c+CD11b+ and CD11c+B220+. Concomitant with the changes in DC numbers, pulmonary DCs production of IL-12p70 upon LPS stimulation was decreased, whereas these cells produced much more IL-10 during the MODS stage compared with DCs from sham mice. The development of a type 1 immune response requires the predominance of type 1 versus type 2 cytokines [11,12]; however, the persistent impairment in IL-12p70 production in MODS lungs appears to result in an imbalance between type 1 and type 2 responses. The defect in IL-12p70 expression by the pulmonary DCs of MODS mice was probably associated with the development of secondary lung injury resulting from immunoparalysis. The histological lung injury and organ dysfunction in zymosan-induced MODS mice coincided with the significantly decreased myeloid and plasmacytoid lung DC numbers and the reduction of DCs expressing MHC-II compared with sham mice.
In contrast, in this study, Flt3L treatment dramatically increased DC production of IL-12p70 and decreased that of IL-10 in MODS mice, probably by altering the type 1/type 2 cytokine profile. Indeed, Flt3L treatment promotes T helper type 1 (Th1) responses and enhances acquired immunity by promoting the expansion of antigen-specific T cells and increasing antigen-specific antibody production. In addition to antigen presentation, DCs express MHC-II that is required for efficient activation of acquired immunity . Expansion of DCs and increased expression of MHC-II should, therefore, have an impact on activating multiple immune functions and possibly eradicate secondary infections and protect MODS animals from further lung injury and organ dysfunction.
Other studies have addressed the effect of Flt3L treatment as an immunotherapeutic option to mitigate against chronic or systemic infections in tuberculosis and Listeria monocytogenes infection models, in which Flt3L treatment has shown diverse effects [31,32]. Toliver-Kinsky et al. found that Flt3L could increase the resistance of mice to Pseudomonas aeruginosa burn wound infection and improved survival rates significantly through both the stimulation of DC production and enhancing DC function . This group also suggested that DC enhancement by Flt3L treatment after burn injury protected against opportunistic infections by promoting local and systemic immune responses to infection .
In the present study, we found a similar loss of lung DCs in the MODS animal model, which supports the concept that severe generalized inflammation results in the systemic loss of DCs, due probably to a deleterious effect on DCs development or increased DCs apoptosis. We observed an altered cytokine production profile in vitro by LPS-stimulated lung DCs from MODS mice, which was related directly to the further lung injury and organ dysfunction due to possible secondary infections because of immunological paralysis. Our results support the possibility of using Flt3L as part of an immunotherapy protocol to enhance acquired immunity induced by lung DCs to secondary infection and tissue damage that targets the lung, as Flt3L promotes the expansion and partial maturation of lung DCs of the myeloid and plasmacytoid subsets. However, additional studies will be necessary to fully characterize and compare the roles of different DC subsets in the altered adaptive immune response in the MODS lung.
Regarding study limitations, to assess lung histological injury we only used a qualitative analysis based on H&E staining of lung tissue sections. In future studies we will use a histological scoring system and immunohistochemical staining for specific markers to provide quantitative assessments of the extent of lung injury and the specific cell types involved. In addition, more precise evaluations of DCs are required as numerous DC subsets may be involved based upon characterizations using multiple markers . There are also the possibilities that secondary infection causes significant lung tissue injury and mortality and that other organ systems are involved. Based on our unpublished data, we have found that Flt3L had protective effects against injuries to other organs, such as the liver, kidney and the gastrointestinal system. These important issues will also need to be addressed in future investigations.
In conclusion, in this study we have demonstrated for the first time that daily administration of the DC growth factor Flt3L for 9 consecutive days after the acute phase of the ZIGI model could attenuate secondary lung tissue damage and decrease the mortality associated with zym-induced MODS in mice. These findings support that Flt3L may be a useful therapy for conditions associated with non-septic shock and is a potential therapeutic agent for patients with MODS.
This study was supported financially in by part by grants from the National Natural Science Foundation (Project no. 81000848) of PR China.