Abstract
Intrahepatic immune cells (IHIC) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. In the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of IHIC. These cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and DNase. The mechanical disruption yielded viable IHIC in considerably greater numbers than those obtained following enzymatic digestion. The IHIC isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which B, T, natural killer (NK), NK T cells, granulocytes and macrophages were the major populations (constituting 37·5%, 16·5%, 12·1%, 7·9%, 7·9% and 7·5% of the total number of cells recovered respectively). The IHIC obtained following enzymatic digestion contained markedly lower numbers of NK T cells (1·8%). The B, T and NK T cells among IHIC isolated employing mechanical disruption were found to be immunocompetent, i.e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin A and α-galactosylceramide respectively) and produced immunoglobulin M and interferon-γ. Thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent IHIC for various types of investigation.
Keywords: enzymatic digestion, flow cytometry, immunophenotyping, intrahepatic immune cells, mechanical disruption
Introduction
The mammalian liver performs a variety of immunological functions, generating immunological tolerance to a large number of dietary antigens received directly from the gastrointestinal tract and, at the same time, responding to blood-borne and dietary pathogens with effective immunological defences [1–4]. Although the intrahepatic immune cells (IHIC) are known to perform these diverse functions [1–5], lack of satisfactory procedures for isolation of these cells has hindered their detailed phenotypic and functional characterization.
Histological analysis of healthy liver, together with studies on isolated cells, have revealed that in both humans and mice IHIC constitute approximately 50% of the non-parenchymal cells (i.e. cells other than hepatocytes) in this organ [4,6]. These IHIC differ in composition and phenotype from the immune cells located in other organs such as the spleen, thymus and lymph nodes. For example, natural killer (NK) cells, NK T cells, γδ T cells and memory T cells are highly enriched in the liver [4,6–9], whereas the proportions of naive T and B lymphocytes have been reported to be relatively small [4,6–9]. Interestingly, both quantitative and functional alterations in IHIC populations occur in response to exposure to microbial pathogens and in connection with the development of immunological tolerance [1,2,4,5,8,10].
Precise and definitive immunophenotypic and functional characterization of IHIC requires isolation of these cells in a viable state, with high yield and a satisfactory degree of purity. Mechanical disruption and/or enzymatic digestion are the two principal procedures employed in the initial phase of the separation of both murine and human IHIC [11–18]. Low cell yields, poor viability and contamination by dead cells and debris are the major problems associated with mechanical disruption [11,13,14,18], while enzymatic digestion, which usually involves collagenase IV and DNase I [12–14], reduces contamination by dead cells and debris, but does not improve cell yields or viability [12–14,19]. Furthermore, enzyme preparations may also digest proteins expressed at the surface of IHIC, as has been observed in the case of murine NK1.1 and its human homologue CD56 [13,17]. Accordingly, the development of more satisfactory approaches to the isolation of IHIC would be of considerable value.
Here, we describe the isolation of murine IHIC following a simple and novel approach to mechanical disruption of the liver, and compare their yields, viability and phenotypes with those obtained following enzymatic digestion. Our procedure results in improved isolation of single cell suspensions of hepatic immune cells that can be subjected to phenotypic analysis by flow cytometry and used for functional in vitro assays of, e.g. cell proliferation and secretion of immunoglobulin (Ig) and cytokines.
Materials and methods
Chemicals
Heparin (sodium salt, grade I-A, from porcine intestinal mucosa), collagenase IV, DNase I, NaN3, ammonium chloride, sodium bicarbonate, 2-mercaptoethanol, bovine serum albumin (BSA), lipopolysaccharide (LPS, Escherichia coli 055:B5) and chemicals for preparing complete phosphate-buffered saline (PBS) and Hanks’ balanced salt solution (HBSS) (including sodium chloride, potassium chloride, dipotassium hydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate) were purchased from Sigma-Aldrich Sweden AB (Stockholm, Sweden). RPMI-1640 medium containing GlutaMAX™-1 and 25 mM HEPES, sodium pyruvate, a solution of penicillin–streptomycin and heat-inactivated fetal calf serum (FCS) were obtained from Invitrogen AB (Stockholm, Sweden). Concanavalin A (ConA) and Percoll™ were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden) and α-galatosylceramide (α-GalCer) from Axxora, LLC (San Diego, CA, USA).
Animals
Six 8-week-old male C57BL/6 (H-2b) mice were obtained from Scanbur AB (Sollentuna, Sweden) and housed in the animal facilities at the Wenner-Gren Institute, Stockholm University, with a 12-h dark/12-h light cycle and access to tap water and standard chow [R70 containing 4·5% fat, 14·5% protein and 60·1% carbohydrate (Lantmännen, Stockholm, Sweden)]ad libitum. The experiments described here were pre-approved by the Northern Stockholm Ethical Committee for Animal Experimentation (N90/07).
Mechanical disruption and enzymatic digestion of the liver and subsequent isolation of IHIC
The mice were killed by inhalation of carbon dioxide, following which the abdomen of each animal was immediately opened and the blood from the heart (approximately 0·7 ml) removed using a 1-ml syringe connected to a 27-G needle and placed into a tube for capillary blood collection (Microtainer BD Bioscience, NJ, USA) containing ethylenediamine tetraacetic acid disodium salt. Thereafter, 5 ml cold PBS (pH 7·2) was injected via the right ventricle of the heart to perfuse the liver until this organ became blanched and swollen. The portal vein was then cut and perfusion with an additional 7 ml PBS in the same manner. Thereafter, the gallbladder was removed and the liver excised carefully from the abdomen.
Isolation of IHIC employing mechanical disruption of the liver was performed as follows: first, this organ was minced into small pieces with surgical scissors and forced gently through a 200 µm-gauge stainless steel mesh using a sterile syringe plunger and the preparation thus obtained was suspended in 50 ml RPMI-1640 medium containing GlutaMAX™-1, 25 mM HEPES and 10% FCS (pH 7·4). In step 2, this suspension was centrifuged at 507 r.p.m. (60 g) with the off-brake setting for 1 min at room temperature. In step 3, the resulting supernatant (45 ml) containing the HIC was transferred to a new tube and centrifuged at 1433 r.p.m. (480 g) with the high-brake setting for 8 min at room temperature. In step 4, the pellet thus obtained was resuspended in 10 ml 37·5% Percoll in HBSS containing 100 U heparin per ml and then centrifuged at 1907 r.p.m. (850 g) with the off-brake setting for 30 min at room temperature. In step 5, this new pellet was resuspended in 2 ml ammonium chloride/Tris-chloride (pH 7·2) (erythrocyte lysing buffer), incubated at room temperature for 5 min, then supplemented with 1 ml FCS and centrifuged subsequently at 1433 r.p.m. (480 g) with the high-brake setting for 8 min at 8°C. Finally, the pellet obtained was resuspended either in 1 ml PBS containing 1% FCS or 0·1% NaN3 (FACS buffer) and subjected to cell surface phenotyping by flow cytometry or in an appropriate volume of cell culture medium for further functional analysis in vitro (see below).
Enzymatic digestion of the liver (according to the method of Huang et al.[20] and subsequent isolation of IHIC was performed as follows: first, a suspension of liver cells was prepared in the same manner as in step 1 described above. In step 2, this suspension was centrifuged at 1500 r.p.m. (528 g) with the high-brake setting for 10 min at 4°C. In step 3, the resulting pellet was resuspended in 10 ml digestion buffer (i.e. collagenase and DNase at concentrations of either 0·2 and 0·02 g/l (method A) or 0·1 and 0·01 g/l (method B), respectively, dissolved in RPMI-1640 medium containing GlutaMAX™-1 and 25 mM HEPES, pH 7·4) and incubated thereafter in a water bath at 37°C with shaking (1 cycle/s) for 40 min at 37°C. In step 4, 30 ml RPMI-1640 medium containing GlutaMAX™-1 and 25 mM HEPES, pH 7·4 was added to the digested suspension and this suspension was allowed to stand on ice for 5 min, after which the top 30 ml were transferred to another tube and centrifuged at 1500 r.p.m. (528 g) with the high-brake setting for 10 min at 4°C. The subsequent steps were identical to those employed in the case of mechanical disruption (see above), with the exception that in the present case, in step 5, the pellet was resuspended in 10 ml 35% Percoll. A flow diagram of the procedures employed for mechanical disruption and enzymatic digestion is presented in Fig. 1.
Fig. 1.
Schematic illustration of the isolation of intrahepatic immune cells (IHIC) from the liver of C57BL/6 mice employing our improved procedure for mechanical disruption or enzymatic digestion.
Assessment of the yield and viability of the isolated IHIC
In connection with the first and final steps of each isolation procedure described above, the numbers of viable IHIC (identified on the basis of their small size, round shape and shiny appearance and assessed as viable on the basis of Trypan blue exclusion) were counted in a haemocytometer. In addition, in order to determine whether either of these procedures enhances the frequency of apoptosis among IHIC, the cells obtained in the final step were first washed with PBS containing calcium, then incubated for 15 min at room temperature with 0·025 µg annexin V conjugated with fluorescein isothiocyanate (FITC) (Apoptosis Detection Kit; R&D Systems Europe, Oxon, UK) in combination with 0·5 µg propidium iodide (PI) in accordance with the manufacturer's instructions, and finally analysed on a fluorescence activated cell sorter (FACSCalibur) flow cytometer (Becton Dickinson, San Jose, CA, USA) (see further below).
Preparation of blood samples for flow cytometry
In order to lyse the erythrocytes present, 100-µl samples of peripheral blood were incubated with 2 ml ammonium chloride-based lysing solution for 10 min at room temperature, following which the suspensions were centrifuged at 900 r.p.m. (200 g) with the high-brake setting for 7 min at 5°C. The resulting pellet was resuspended in 0·5 ml PBS containing 1% FCS and 0·1% NaN3 for flow cytometric analysis of cell surface phenotypes (see below).
Immunofluorescent staining and flow cytometric analysis
Immunophenotyping of IHIC and peripheral blood cells was performed using a panel of antibodies directed against the appropriated murine cell surface antigens. The antibodies employed were the following: anti-CD45 labelled with (-)fluorescein isothiocyanate (FITC) (clone 30F11), anti-CD4-FITC (clone RM4-4), anti-CD19-FITC (clone 1D3), anti-γδ T cell receptor-FITC (clone GL3), anti-CD11b-FITC (clone M1/70), anti-NK1·1-FITC (clone PK136), anti-NK1·1 labelled with (-)R-phycoerythrin (PE) (clone PK136), anti T cell receptor β chain-PE (clone H57-597), anti-Gr1-PE (clone H57-597) and anti-CD8a labelled with peridinin chlorophyll protein (PerCP) (clone 53-6·7). IHIC or peripheral white blood cells (105 in both cases) suspended in 50 µl FACS buffer were added to each individual well of 96-well round-bottomed tissue culture plates (Corning, NY, USA) and then incubated with antibodies directed towards various cell surface antigens and in the dark on ice for 30 min, in accordance with the manufacturer's recommendations (BD PharMingen, San Diego, CA, USA). Subsequently, the cells were washed by centrifugation at 200 g for 10 min, resuspended in 400 µl FACS buffer and analysed utilizing a single-laser FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 15-mW, air-cooled 488 nm argon-ion laser. The signals emitted by FITC, PE and PerCP were detected at 530, 575 and 670 nm respectively. For each sample, the data from 10 000 events (individual cells) were collected and analysed employing CellQuest Software.
Preparation of cell suspensions from the spleen
Spleens were dissected out aseptically and thereafter teased apart gently with forceps in RPMI-1640 medium containing GlutaMax™-1 supplemented with 15 mM HEPES, 100 IU penicillin, 100 µg streptomycin per ml and 0·02% (w/v) sodium bicarbonate (pH 7·4). The splenocytes thus obtained were washed twice with culture medium, counted and employed as a positive control in the functional assays (see below).
Cell culture
The IHIC isolated following mechanical disruption of the liver were resuspended in RPMI-1640 medium containing GlutaMax™-1 and 25 mM HEPES (pH 7·4) and supplemented with 15% FCS, 100 IU penicillin and 100 µg streptomycin per ml, 0·5 µg/ml fungizone, 1 mM sodium pyruvate, 0·02% (w/v) sodium bicarbonate and 5 × 10−5 M 2-mercaptoethanol. After adjusting the concentration to 9 × 106 viable cells (as determined by Trypan blue exclusion) per ml (containing approximately 3·3 × 106 B cells, 1·4 × 106 T cells and 0·7 × 106 NK T cells), each suspension was cultured in triplicate (100 µl per well) in 96-well flat-bottomed culture plates (Costar, Corning Incorporated). Thereafter, 100 µl of complete RPMI-1640 medium containing either LPS (25 µg/ml), ConA (3 µg/ml) or α-GalCer (100 ng/ml) was added to each well and the plates incubated for 72 or 90 h (the optimal periods for the proliferation of B, T and NK T cells and for cytokine production by T and NK T cells and antibody production by B cells respectively [21–24], at 37°C under a humidified atmosphere containing 5% CO2. Identical conditions were used to culture the splenocytes, with the exception that in this case a concentration of 3 × 106 cells/ml was utilized.
Evaluation of cell proliferation
Cell proliferation was evaluated employing the methyl thiazol tetrazolium (MTT)-based [25] kit (Sigma-Aldrich Sweden AB, Stockholm, Sweden) in accordance with the manufacturer's instructions. Briefly, 72 h after stimulation with LPS, ConA or α-GalCer, 20 µl MTT was added to each well and incubation continued for another 4 h at 37°C under a humidified atmosphere with 5% CO2. Subsequently, the formazan crystals formed as a result of mitochondrial dehydrogenase activity were solubilized and the difference in absorbance at 570 and 690 nm determined 15 min later utilizing a microplate reader (Molecular Devices Corporation Orleans Drive, Sunnyvale, CA, USA). The stimulation index = the absorbance obtained with LPS-activated cultures divided by the corresponding value for non-stimulated cultures.
Quantitation of IgM
The media from the cell cultures stimulated with LPS were collected after 90 h of incubation and their contents of IgM detemined with a sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, for this purpose each well of 96-well, flat-bottomed micro-ELISA plates (Nunc A/S, Roskilde, Denmark) was coated with goat anti-mouse Ig (1 µg/ml) (Southern Biotechnology, Birmingham, AL, USA) overnight at 4°C, following which the remaining free binding sites were blocked by incubation for 2 h at room temperature with BSA (1%, v/v) dissolved in PBS. After washing these plates three times with PBS-Tween, 100 µl of cell culture medium was added to each well and the plates incubated subsequently at room temperature for 4 h, washed four times with PBS-Tween, then incubated with alkaline phosphatase-labelled goat anti-mouse IgM (at a dilution of 1/2000; Southern Biotechnology) for 2 h at room temperature and thereafter washed three more times. Next, each well was incubated for 30 min at room temperature with a solution of the phosphatase substrate p-nitrophenyl phosphate (disodium salt hexahydrate; Sigma) and the absorbance at 405 nm finally determined on a microplate reader. The concentrations of IgM in the media were assessed by comparison with a calibration curve generated using an internal standard (Southern Biotechnology).
Quantitation of interferon-γ
The media from the cell cultures stimulated with ConA or α-GalCer were collected after 72 h of incubation and their content of interferon (IFN)-γ detemined with a commercially available ELISA kit for murine IFN-γ (Mabtech AB, Stockholm, Sweden). The ELISA was performed according to the manufacturer's instructions and optical density determined at 405 nm. In all cases, a standard curve was constructed from standards provided by the manufacturer.
Expression and statistical analysis of the data
All data are expressed as means ± standard error. Differences between the numbers and phenotypes of the cells isolated following the improved mechanical disruption and enzymatic digestion procedures A and B were analysed for statistical significance using analysis of variance. Differences in cell proliferation and the production of IgM and IFN-γ by IHIC activated in vitro with or without LPS, ConA or α-GalCer and differences between the blood and IHIC cell populations were analysed for statistical significance employing the Mann–Whitney U-test. All these statistical analyses were performed utilizing WinSTAT software (R. Fitch Software, Medina AB, Vänerborg, Sweden).
Results
Comparison of cell yields and viability obtained employing mechanical disruption and enzymatic digestion
Pilot experiments under a number of different conditions, including the use of different temperatures, Percoll concentrations and centrifugal forces, revealed that the conditions described in detail in the Materials and methods section and illustrated schematically in Fig. 1 provide maximal cell yields with optimal viability.
The initial teasing apart and passage of the liver through a 200-µm sieve, the common first step in both the mechanical disruption and enzymatic procedures, yielded suspensions containing 5·9–8·3 × 106 immune cells per liver (from a total of 12 animals examined in three independent experiments). In general, the time required to isolate IHIC by the improved procedure for mechanical disruption (50 min) was shorter than in the case of enzymatic digestion method (106 min) (Fig. 1). Moreover, the mechanical disruption resulted in appreciably higher cell yields (64%) than those obtained by either procedure A (10·1%) or B (52%) involving enzymatic digestion (n = 18 in two independent experiments; Fig. 2).
Fig. 2.
Enzymatic digestion with collagenase and DNase results in considerably lower recovery of intrahepatic immune cells (IHIC) than that obtained with mechanical disruption. Intrahepatic immune cells from the liver of C57BL/6 male mice were isolated employing either our improved procedure for mechanical disruption (solid bar) or enzymatic digestion (grey bars): (a) collagenase and DNase at concentrations of 0·2 and 0·02 g/l respectively; (b) collagenase and DNase at concentrations of 0·1 and 0·01 g/l respectively), as described in detail in the Materials and methods and illustrated in Fig. 1. The total numbers of viable IHIC were evaluated subsequently on the basis of Trypan blue exclusion. The values shown are means ± standard errors (n = 18 and 12 mice) for mechanical disruption and enzymatic digestion respectively. *P < 0·05 and **P < 0·01 as determined by analysis of variance.
With both types of procedure, > 90% of the cells obtained were viable (i.e. excluded Trypan blue). Furthermore, as determined by staining with annexin V-FITC and PI and subsequent analysis by flow cytometry, there was no difference in the numbers of apoptotic plus necrotic cells in the final cell suspensions (5 ± 1·5% in the case of mechanical disruption versus 3 ± 0·8% with enzymatic digestion; n = 12 in two independent experiments). Thus, our procedure for mechanical disruption allows recovery of a considerably larger number of viable IHIC from murine liver.
Immunophenotypic analysis of IHIC isolated with the improved procedure for mechanical disruption or following enzymatic digestion
Phenotypic characterization of the purified IHIC by immunofluorescence staining and flow cytometry revealed that with both of these procedures more than 97% of the gated cells expressed CD45, a pan-leucocytic marker present on the surface of all cells derived from the bone marrow, with the exception of erythrocytes and platelets [26] (Table 1). Subsequent immunophenotypic analysis of subpopulations of the CD45+ cells revealed that the IHIC isolated following the mechanical disruption were heterogeneous in composition, containing both innate and adaptive immune cells of which B, T, NK and NK T cells, granulocytes, macrophages, γδ T and so-called myeloid suppressor cells, which express CD11b and Gr1 cell surface markers [27], were the major populations (Table 1).
Table 1.
Phenotypic analysis of intrahepatic immune cells isolated following modified mechanical disruption or enzymatic digestion.
| Enzymatic digestion |
|||
|---|---|---|---|
| Mechanical disruption | Method Aa | Method Ba | |
| Subpopulation (surface marker) | Proportion of the total number of cells recovered (%)b | ||
| All leucocytes (CD45+) | 98·0 ± 1·1 | 98·8 ± 0·4 | 98·4 ± 0·5 |
| NK cells (NK1·1+TCR−) | 12·1 ± 0·7 | 13·2 ± 0·3 | 12·7 ± 0·1 |
| NK T cells (NK1·1+TCR+) | 7·9 ± 0·1**c | 1·8 ± 0·2 | 1·3 ± 0·2 |
| Granulocytes (Gr1+) | 7·9 ± 0·6 | 8·7 ± 0·3 | 11·3 ± 0·8**d |
| Macrophages (CD11b+) | 7·5 ± 0·7 | 8·4 ± 0·8 | 9·2 ± 0·8 |
| γδ T cells (γδ+) | 5·4 ± 0·6*e | 3·4 ± 0·2 | 4·8 ± 0·7 |
| Myeloid suppressor cells (CD11b+Gr1+) | 3·1 ± 0·4 | 3·3 ± 0·2 | 4·7 ± 0·5*f |
| B cells (CD19+) | 37·5 ± 0·7 | 44·1 ± 1·0*g | 36·1 ± 2·3 |
| Helper T cells (CD4+ NK1·1−) | 9·0 ± 1·7 | 7·0 ± 0·8 | 8·6 ± 0·4 |
| Cytotoxic T cells (CD8+ NK1·1−) | 7·6 ± 0·5 | 8·4 ± 0·6 | 9·1 ± 0·7 |
aThe concentrations of collagenase and DNase (w/v) used in the enzymatic digestion procedures A and B are shown in Fig. 1. b All values presented are means ± standard errors for nine animals. c** P < 0·01 for mechanical disruption versus both enzymatic digestion procedures (A and B) as analysed by analysis of variance (anova). d,f** P < 0·01 and *P < 0·05, respectively, for enzymatic digestion procedure (B) versus both mechanical disruption and enzymatic digestion procedure (A), as analysed by anova. e* P < 0·01 for mechanical disruption versus enzymatic digestion procedure (A) as analysed by anova. g* P < 0·05 for the enzymatic digestion procedure (A) versus both mechanical disruption and enzymatic digestion procedure (B), as analysed by anova. NK, natural killer cells; NK T, natural killer T cells; TCR, T cell receptor.
The composition of IHIC obtained with enzymatic digestion was significantly different. For instance, treatment with collagenase and DNase at concentrations of 0·2 and 0·02 g/l, respectively (method A), resulted in a significantly larger percentage of B cells but, at the same time, considerably lower proportions of NK T and γδ T cells (Table 1). Following incubation of the liver tissue with lower concentrations of collagenase and DNase (method B), the relative percentages of granulocytes and myeloid suppressor cells were higher, the percentages of B and γδ T cells unaltered and the proportion of NK T cells recovered still significantly lower than with mechanical disruption (Table 1). These findings indicate that murine intrahepatic NK T cells are highly sensitive to treatment with collagenase and DNase.
We also determined the interassay variation for our procedure for mechanical disruption with regard to the total number of cells recovered, as well as the populations of hepatic immune cells of different phenotypes, and compared this interassay variation with that for enzymatic digestion (method A) employing coefficients of variation (CV). For both procedures, the interassay CV values for the total number of cells recovered, as well as for the numbers of CD45+, NK T, B and helper T cells were low and comparable (1·2–4%). On the other hand, these interassay CV values for granulocytes and NK cells were lower with enzymatic digestion (4% and 6% respectively) than mechanical disruption (10·5% and 14% respectively). With both procedures, the interassay CV values for cytotoxic T, γδ T and myeloid suppressor cells were relatively high (12·5–20%).
The compositions of immune cell populations in the liver and peripheral blood differ considerably
In order to minimize possible contamination of the IHIC by circulating immune cells, the liver was perfused thoroughly prior to initiation of both isolation procedures. Moreover, we also found that the composition of the IHIC prepared employing mechanical disruption differed considerably from that of the circulating immune cells. The immune cells present in peripheral blood contained a much higher percentage of CD19+ cells, while the relative proportions of NK T and NK cells were higher in the liver (Table 1versusTable 2). These observations confirm that there is relatively little contamination of these IHIC by blood leukocytes.
Table 2.
Subpopulations of immune cells in the peripheral blood of male C57BL/6 mice.
| Subpopulation (surface marker) | Immune cells in the peripheral blood (%)† |
|---|---|
| NK cells (NK1·1+/TCR−) | 5·9 ± 0·8*‡ |
| NK T cells (NK1·1+/TCR+) | 1·2 ± 0·1**‡ |
| Granulocytes (Gr1+) | 5·8 ± 0·3 |
| Macrophage/monocytes (CD11b+) | 6·4 ± 0·6 |
| γδ T cells (γδ+) | 6·9 ± 0·1 |
| Myeloid suppressor cells (CD11b+/Gr1+) | 9·6 ± 0·8*‡ |
| B cells (CD19+) | 50 ± 2**‡ |
| Helper T cells (CD4+/NK1·1−) | 11·7 ± 0·6 |
| Cytotoxic T cells (CD3+/CD8+) | 4·2 ± 0·3*‡ |
All values are the means ± standard errors for three animals.
P < 0·01 compared with the corresponding cell population in intrahepatic immune cells isolated employing our improved procedure for mechanical disruption (Table 1), as analysed by the Mann–Whitney U-test. NK, natural killer cells; NK T, natural killer T cells; TCR, T cell receptor.
Functional analysis of intrahepatic B, T and NK T cells isolated following the improved mechanical disruption
In order to evaluate whether IHIC obtained with our improved procedure for mechanical disruption were functionally intact, we examined in vitro the proliferative responses of the B, T and NK T cells as well as the production of IgM by B cells and IFN-γ by T and NK T cells, upon exposure to their specific activating stimuli (i.e. LPS, ConA and α-GalCer respectively). As shown in Fig. 3a,b, in response to LPS, hepatic B cells proliferated and produced significant amounts of IgM (although both these responses were less pronounced than those observed with splenic B cells, the positive control). Similarly, despite their lower proliferative response in comparison with splenocytes, T cells present in the IHIC responded to ConA by producing high levels of IFN-γ (Fig. 4a,b). Moreover, hepatic, but not splenic NK T cells responded to α-GalCer by synthesizing significant amounts of IFN-γ (Fig. 5a,b). These observations demonstrate that these three cell populations in IHIC isolated following the improved mechanical disruption are functionally immunocompetent.
Fig. 3.
The B cells among the intrahepatic immune cells (IHIC) isolated employing our improved procedure for mechanical disruption are functionally immunocompetent. IHIC were isolated from the liver of C57BL/6 male mice employing mechanical disruption (see the Materials and methods and Fig. 1 for further details) and, as a positive control, splenocytes were prepared from these same animals simply by teasing apart the spleen. Both IHIC (solid bars) and splenocytes (grey bars) were cultured in the absence or presence of lipopolysaccharide (LPS; 25 µg/ml) for either 72 (a) or 90 h (b). (a) Cell proliferation was assessed on the basis of mitochondrial reduction of methylthiazol tetrazolium (MTT), as described in the Materials and methods, and the stimulation index (SI) ± standard errors (s.e.) (n = 3) calculated as the absorbance values for LPS-activated cultures divided by the absorbance values for non-stimulated cultures. (b) Immunoglobulin (Ig)M concentrations in the cell culture supernatants were determined with a sandwich enzyme-linked immunosorbent assay (ELISA). The values shown are means ± s.e. (n = 3) and the differences were analysed for statistical significance employing the U-test. **P < 0·01 compared with the level of IgM present in the media of cell cultures from which LPS was absent.
Fig. 4.
The T cells among the intrahepatic immune cells (IHIC) isolated employing the improved procedure for mechanical disruption are functionally immunocompetent. IHIC were isolated from the liver of C57BL/6 male mice employing mechanical disruption (see the Materials and methods and Fig. 1 for further details) and, as a positive control, splenocytes were prepared from these same animals simply by teasing apart the spleen. Both IHIC (solid bars) and splenocytes (grey bars) were cultured in the absence or presence of concanavalin A (ConA; 3 µg/ml) for 72 h. (a) Cell proliferation was assessed on the basis of mitochondrial reduction of methylthiazol tetrazolium (MTT), as described in the Materials and methods, and the stimulation index (SI) values ± standard errors (s.e.) (n = 3) calculated as the absorbance values for ConA-activated cultures divided by the absorbance values for non-stimulated cell cultures. (b) Interferon (IFN)-γ concentrations in the cell culture media were detemined with a sandwich enzyme-linked immunosorbent assay (ELISA). The values shown are means ± s.e. (n = 3) and the differences were analysed for statistical significance employing the U-test. **P < 0·01 compared with the level of IFN-γ present in the media of cell cultures from which ConA was absent.
Fig. 5.
The natural killer T (NK T) cells among the intrahepatic immune cells (IHIC) isolated employing the improved procedure for mechanical disruption are functionally immunocompetent. IHIC were isolated from the liver of C57BL/6 male mice employing mechanical disruption (see the Materials and methods and Fig. 1 for further details) and, as a control, splenocytes were prepared from these same animals simply by teasing apart the spleen. Both IHIC (solid bars) and splenocytes (grey bars) were cultured in the absence or presence α-galatosylceramide (α-GalCer; 100 ng/ml) for 72 h. (a) Cell proliferation was assessed on the basis of mitochondrial reduction of methylthiazol tetrazolium (MTT), as described in the Materials and methods section, and the stimulation index (SI) values ± standard errors (s.e.) (n = 3) calculated as the absorbance values for α-GalCer-activated cultures divided by the absorbance values for non-stimulated cell cultures. (b) Interferon (IFN)-γ concentrations in the cell culture media were detemined with a sandwich enzyme-linked immunosorbent assay (ELISA). The values shown are means ± s.e. (n = 3) and the differences analysed for statistical significance employing the U-test. *P < 0·05 compared with the level of IFN-γ present in the media of cell cultures from which α-GalCer was absent.
Discussion
In this study, we describe a modified procedure for mechanical disruption of mouse liver that provides a considerably higher yield of viable IHIC than those obtained employing enzymatic digestion, according to published procedures [20,28,29]. Furthermore, the present procedure for mechanical disruption is also more effective than another reported mechanical procedure described by Dong et al.[13]. These investigators first disrupted the perfused liver, passed the resulting suspension through a 200-gauge stainless steel mesh and then suspended the filtrate in RPMI-1640 medium containing FCS. Thereafter, this liver cell suspension was centrifuged at 526 g and the pellet obtained resuspended in 40% Percoll solution containing 100 U/ml heparin and then loaded onto the solution of 70% Percoll and centrifuged at 935 g for 20 min at room temperature. In the final steps, the cells were aspirated from the Percoll interface, harvested by centrifugation and washed twice with HBSS containing FCS [13]. With this procedure, approximately 2·6 × 106 immune cells were recovered from each liver [13], a number significantly lower than the recovery documented here following our mechanical disruption (3·8 × 106 cell/liver). As described below, a more striking difference is that the mechanical approach described by Dong et al.[13] results in the recovery of certain, but not all hepatic immune cells. Thus, the efficacy, simplicity and low cost of the mechanical procedure developed here represent advantages over previously described methods for the isolation of IHIC.
The present observation that treatment of mouse liver with collagenase and DNase at higher (0·2 and 0·02 g/l respectively), but not lower (0·1 and 0·01 g/l) concentrations, results in poor recovery of IHIC indicates, as observed by others [13,14], that extensive enzymatic digestion exerts deleterious effects on these cells. Thus, careful optimization of enzyme concentrations is required when such digestion is utilized for the isolation of IHIC.
By immunostaining with a panel of antibodies and subsequent flow cytometric analysis, we confirm here that murine liver normally contains cells belonging to both the innate (macrophages, granulocytes, myeloid suppressor cells, NK, NK T and γδ T cells, constituting altogether 44% of the IHIC isolated) and the adaptive (B, helper T and cytotoxic T cells; 54% of the total IHIC) branches of the immune system. Moreover, the relative sizes of certain of the populations of immune cells present in the liver and peripheral blood differ. For example, cells involved in innate immunity, and in particular NK and NK T cells, are less common in the peripheral blood (where they represent 5·9% and 1·2%, respectively, of all the immune cells present) than in the liver (12·1% and 7·9% respectively). Clearly, a distinct pattern of immune cell populations is normally resident in the liver of mice, reflecting the specific active immunological responses mediated by this organ.
Flow cytometric analysis of IHIC immunostained following isolation revealed here that treatment with collagenase and DNase alters the composition of these cells in a concentration-dependent manner. Of particular interest in this respect is that, irrespective of the concentration used, treatment with these enzymes markedly reduces the recovery of NK T cells (to 1·3% of the total cell number), in agreement with an earlier report [13]. Thus, enzymatic digestion appears to be an inappropriate approach for the isolation and characterization of murine hepatic NK T cells.
The finding that the interassay CV values for several parameters, including total number of recovered cells and numbers of NK T, B and helper T cells, with our procedure for mechanical disruption are low and comparable to the corresponding values for enzymatic digestion indicate satisfactory reproducibility. However, the observation that the interassay CV values for granulocytes and NK cells are higher in the case of our mechanical disruption than for enzymatic digestion suggests that disruption of the liver into finer pieces might improve the reproducibility of our procedure with respect to these parameters.
Although the liver is known to be the exclusive site of B cell lymphopoiesis during embryonic development [30,31], the presence of these cells in the adult murine liver is either underestimated or not mentioned in most studies in this area or, sometimes, these cells are even eliminated selectively employing a specific procedure [4,6–9,13]. For instance, the mechanical disruption approach employed by Dong et al. [13] yields T (39%), NK (13%), NK T (20%) and γδ T cells (14%), but no B cells. In contrast, utilization of our improved procedure for mechanical disruption yields IHIC containing 37–44% B cells, suggesting that these cells play a significant role in hepatic immune responses to foreign antigens. This suggestion is supported by the observation that hepatic B cells play a crucial role in the pathogenesis of chemically induced liver fibrosis [32].
The demonstration that IHIC isolated by our protocol respond to specific activators of B, T and NK T cells in vitro with both proliferation and the production of IgM and IFN-γ clearly imply that these cells are functionally immunocompetent. Moreover, the finding that in response to α-GalCer, IHIC, but not splenocytes produce high levels of IFN-γ support the observation that NK T cells are highly enriched in liver [4,6–9]. Thus, our improved procedure for mechanical disruption is suitable not only for phenotypic but also for functional analysis of IHIC.
In summary, we have developed a simple procedure for the mechanical disruption of mouse liver that allows the isolation of IHIC in yields considerably higher than those achieved by other reported procedures. In addition, the cells thus isolated are amenable to culturing and are functionally immunocompetent. This procedure should prove to be highly valuable in connection with attempts to elucidate the hepatic mechanisms underlying the development of immunological tolerance, as well as other immunological responses mediated by the liver. Furthermore, it will be of considerable interest to examine changes in the composition and activities of IHIC in response to environmental agents such as pathogens and xenobiotics.
Acknowledgments
This study was financed by an unrestricted grant from the 3M Company (St Paul, Minnesota, USA).
References
- 1.Knolle PA, Gerken G. Local control of the immune response in the liver. Immunol Rev. 2000;174:21–34. doi: 10.1034/j.1600-0528.2002.017408.x. [DOI] [PubMed] [Google Scholar]
- 2.Crispe IN. Hepatic T cells and liver tolerance. Nat Rev Immunol. 2003;3:51–62. doi: 10.1038/nri981. [DOI] [PubMed] [Google Scholar]
- 3.Bowen DG, McCaughan GW, Bertolino P. Intrahepatic immunity: a tale of two sites? Trends Immunol. 2005;26:512–7. doi: 10.1016/j.it.2005.08.005. [DOI] [PubMed] [Google Scholar]
- 4.Racanelli V, Rehermann B. The liver as an immunological organ. Hepatology. 2006;43:S54–62. doi: 10.1002/hep.21060. [DOI] [PubMed] [Google Scholar]
- 5.Parker GA, Picut CA. Liver immunobiology. Toxicol Pathol. 2005;33:52–62. doi: 10.1080/01926230590522365. [DOI] [PubMed] [Google Scholar]
- 6.Klugewitz K, Adams DH, Emoto M, Eulenburg K, Hamann A. The composition of intrahepatic lymphocytes: shaped by selective recruitment? Trends Immunol. 2004;25:590–4. doi: 10.1016/j.it.2004.09.006. [DOI] [PubMed] [Google Scholar]
- 7.Crispe IN, Mehal WZ. Strange brew: T cells in the liver. Immunol Today. 1996;17:522–5. doi: 10.1016/s0167-5699(96)80906-6. [DOI] [PubMed] [Google Scholar]
- 8.Li Z, Diehl AM. Innate immunity in the liver. Curr Opin Gastroenterol. 2003;19:565–71. doi: 10.1097/00001574-200311000-00009. [DOI] [PubMed] [Google Scholar]
- 9.Exley MA, Koziel MJ. To be or not to be NKT: natural killer T cells in the liver. Hepatology. 2004;40:1033–40. doi: 10.1002/hep.20433. [DOI] [PubMed] [Google Scholar]
- 10.Seki S, Habu Y, Kawamura T, et al. The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses. Immunol Rev. 2000;174:35–46. doi: 10.1034/j.1600-0528.2002.017404.x. [DOI] [PubMed] [Google Scholar]
- 11.Goossens PL, Jouin H, Marchal G, Milon G. Isolation and flow cytometric analysis of the free lymphomyeloid cells present in murine liver. J Immunol Methods. 1990;132:137–44. doi: 10.1016/0022-1759(90)90407-m. [DOI] [PubMed] [Google Scholar]
- 12.Trobonjaca Z, Kroger A, Stober D, et al. Activating immunity in the liver. II. IFN-beta attenuates NK cell-dependent liver injury triggered by liver NKT cell activation. J Immunol. 2002;168:3763–70. doi: 10.4049/jimmunol.168.8.3763. [DOI] [PubMed] [Google Scholar]
- 13.Dong ZJ, Wei HM, Sun R, Tian ZG, Gao B. Isolation of murine hepatic lymphocytes using mechanical dissection for phenotypic and functional analysis of NK1.1+ cells. World J Gastroenterol. 2004;10:1928–33. doi: 10.3748/wjg.v10.i13.1928. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Zhang JH, Dong ZJ, Zhou RB, Luo de M, Wei HM, Tian ZG. Isolation of lymphocytes and their innate immune characterizations from liver, intestine, lung and uterus. Cell Mol Immunol. 2005;2:271–80. [PubMed] [Google Scholar]
- 15.Hata K, Zhang XR, Iwatsuki S, Van Thiel DH, Herberman RB, Whiteside TL. Isolation, phenotyping, and functional analysis of lymphocytes from human liver. Clin Immunol Immunopathol. 1990;56:401–19. doi: 10.1016/0090-1229(90)90160-r. [DOI] [PubMed] [Google Scholar]
- 16.Li XM, Jeffers LJ, Reddy KR, et al. Immunophenotyping of lymphocytes in liver tissue of patients with chronic liver diseases by flow cytometry. Hepatology. 1991;14:121–7. doi: 10.1002/hep.1840140120. [DOI] [PubMed] [Google Scholar]
- 17.Curry MP, Norris S, Golden-Mason L, et al. Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterization. J Immunol Methods. 2000;242:21–31. doi: 10.1016/s0022-1759(00)00204-0. [DOI] [PubMed] [Google Scholar]
- 18.Morsy MA, Norman PJ, Mitry R, Rela M, Heaton ND, Vaughan RW. Isolation, purification and flow cytometric analysis of human intrahepatic lymphocytes using an improved technique. Lab Invest. 2005;85:285–96. doi: 10.1038/labinvest.3700219. [DOI] [PubMed] [Google Scholar]
- 19.Wiltrout RH, Pilaro AM, Gruys ME, et al. Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow. J Immunol. 1989;143:372–8. [PubMed] [Google Scholar]
- 20.Huang L, Soldevila G, Leeker M, Flavell R, Crispe IN. The liver eliminates T cells undergoing antigen-triggered apoptosis in vivo. Immunity. 1994;1:741–9. doi: 10.1016/s1074-7613(94)80016-2. [DOI] [PubMed] [Google Scholar]
- 21.Andersson J, Sjoberg O, Moller G. Induction of immunoglobulin and antibody synthesis in vitro by lipopolysaccharides. Eur J Immunol. 1972;2:349–53. doi: 10.1002/eji.1830020410. [DOI] [PubMed] [Google Scholar]
- 22.Peavy DL, Adler WH, Smith RT. The mitogenic effects of endotoxin and staphylococcal enterotoxin B on mouse spleen cells and human peripheral lymphocytes. J Immunol. 1970;105:1453–8. [PubMed] [Google Scholar]
- 23.Andersson J, Moller G, Sjoberg O. Selective induction of DNA synthesis in T and B lymphocytes. Cell Immunol. 1972;4:381–93. doi: 10.1016/0008-8749(72)90040-8. [DOI] [PubMed] [Google Scholar]
- 24.Yu KO, Im JS, Molano A, et al. Modulation of CD1d-restricted NKT cell responses by using N-acyl variants of alpha-galactosylceramides. Proc Natl Acad Sci USA. 2005;102:3383–8. doi: 10.1073/pnas.0407488102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
- 26.Thomas ML. The leukocyte common antigen family. Annu Rev Immunol. 1989;7:339–69. doi: 10.1146/annurev.iy.07.040189.002011. [DOI] [PubMed] [Google Scholar]
- 27.Serafini P, Borrello I, Bronte V. Myeloid suppressor cells in cancer: recruitment, phenotype, properties, and mechanisms of immune suppression. Semin Cancer Biol. 2006;16:53–65. doi: 10.1016/j.semcancer.2005.07.005. [DOI] [PubMed] [Google Scholar]
- 28.Huang L, Sye K, Crispe IN. Proliferation and apoptosis of B220+CD4–CD8–TCR alpha beta intermediate T cells in the liver of normal adult mice: implication for lpr pathogenesis. Int Immunol. 1994;6:533–40. doi: 10.1093/intimm/6.4.533. [DOI] [PubMed] [Google Scholar]
- 29.Klugewitz K, Blumenthal-Barby F, Schrage A, Knolle PA, Hamann A, Crispe IN. Immunomodulatory effects of the liver: deletion of activated CD4+ effector cells and suppression of IFN-gamma-producing cells after intravenous protein immunization. J Immunol. 2002;169:2407–13. doi: 10.4049/jimmunol.169.5.2407. [DOI] [PubMed] [Google Scholar]
- 30.Hardy RR, Hayakawa K. B cell development pathways. Annu Rev Immunol. 2001;19:595–621. doi: 10.1146/annurev.immunol.19.1.595. [DOI] [PubMed] [Google Scholar]
- 31.Hardy RR. B-1 B cell development. J Immunol. 2006;177:2749–54. doi: 10.4049/jimmunol.177.5.2749. [DOI] [PubMed] [Google Scholar]
- 32.Novobrantseva TI, Majeau GR, Amatucci A, et al. Attenuated liver fibrosis in the absence of B cells. J Clin Invest. 2005;115:3072–82. doi: 10.1172/JCI24798. [DOI] [PMC free article] [PubMed] [Google Scholar]