Published online before print January 2, 2004
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German Diabetes Research Institute at the Heinrich-Heine-University Düsseldorf, Germany
1Correspondence: German Diabetes Research Institute, Clinical Department, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany. E-mail: martin{at}ddfi.uni-duesseldorf.de
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as a potential analysis parameter. As the MMLC assay is based on the mutual interaction of cells of the innate and adaptive immunity, it enables the monitoring of cytokine release under almost physiological conditions and might be of interest for the characterization of known and novel drugs concerning their immunomodulatory potency.
Key Words: immunomodulation • immune mediators • LPS • adaptive immunity
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However, the development of a broader panel of immunomodulatory drugs depends on the availability of simple but valid and reliable assay systems. The majority of assay systems uses cells of the innate-immune system such as macrophages and dendritic cells or T cell clones belonging to the adaptive branch of the immune system. In these cell lines as well as in isolated human peripheral blood mononuclear cells (PBMC), strong and/or unphysiological stimuli such as phytohemagglutinin or anti-CD3 antibodies are required to induce measurable responses. Whereas these approaches reveal details of molecular mechanisms of interaction between drugs and specific cell types, the aim of this project was the establishment and analysis of an in vitro test system that enables the screening for substances with immunomodulatory properties under more physiological conditions, allowing for mutual interactions between innate- and adaptive-immune responses.
To establish a valid test system for the analysis of immunomodulatory properties of a wide range of drugs, we combined antigen-presenting cell (APC)-mediated and lipopolysaccharide (LPS)-mediated stimuli to activate innate and adaptive immunity simultaneously in a modified mixed lymphocyte culture (MMLC). We analyze the molecular mechanism of interferon- (IFN-) induction and demonstrate the feasibility of the MMLC system for the characterization of immunomodulation by cytokines and other therapeutically interesting substances.
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MMLC culture conditions and reagents
For allogeneic cell activation, we used two-way MLC. PBMC from different healthy adult blood donors were mixed in equal numbers (5x105 from each donor) and cultured at 450 µl/well in pyrogen-free, 48-well culture plates (Falcon, Heidelberg, Germany). LPS from Escherichia coli 026:B6 (Sigma, Taufkirchen, Germany), recombinant cytokines, and antibodies were added to a final volume of 500 µl. We incubated triplicate cultures under identical conditions without change of medium for up to 5 days at 37°C in a humidified 5% CO2 atmosphere. The cell viability at the end of the incubation period was shown to exceed 90% by the trypan blue dye exclusion test. Culture supernatants were harvested and stored in aliquots at −20°C until cytokine determination.
Human recombinant IL-12 and IL-4 and mouse anti-human IL-12 monoclonal antibody (mAb) were obtained from R&D Systems (Wiesbaden, Germany). Human recombinant IL-18 and mouse anti-human IL-18 mAb were purchased from MBL (Nagoya, Japan). All mAb were specific for the appropriate cytokine without cross-reactivity with other human cytokines. Hydrocortisone was obtained from Amersham Pharmacia Biotech.
Cytokine enzyme-linked immunosorbent assay (ELISA)
The IFN- ELISA was based on a matched mAb pair purchased from Endogen (Woburn, MA) and human recombinant IFN- protein (PharMingen, Heidelberg, Germany) as standard. The detection limit was 4 pg/ml.
Thymidine proliferation assay
For proliferation analysis, cells were incubated in round-bottom plates at a final concentration of 1 x 106 cells/ml. LPS was added in 15 µl to a final concentration of 0.1 ng/ml and a total volume of 150 µl. After 5 days of incubation, each well was pulsed with [3H] thymidine (TRA120 obtained from Amersham Pharmacia Biotech) corresponding to 1 µCi per well in a volume of 50 µl. After 18 h, cells were harvested, washed, transferred to glass fiber membranes (Canberra Packard, Schwadorf, Austria), and counted in scintillation mix (Canberra Packard) in an imaging reader (Canberra Packard).
RNA isolation and reverse transcriptase (RT) reaction
Total RNA from PBMC was stabilized and isolated using the PAXgeneTM blood RNA system (PreAnalytix, Hilden, Germany) according to the manufacturer’s protocol. RT was performed using Ominscript RT kit from Qiagen (Valencia, CA). In brief, the mixture contained 4 µl 10x RT buffer, 0.5 mM deoxy-unspecified nucleoside 5'-triphosphate, 20 U RNase inhibitor (Promega, Mannheim, Germany), 8 U Omniscript RT, 1 µM oligo-dT primer (Roche, Mannheim, Germany), 20 µl sample RNA, and RNase-free water to a total volume of 40 µl. cDNA was aliquoted and stored at −20°C until use.
Quantitative cytokine mRNA analysis
The quantification of cytokine mRNA molecules was performed on an ABI PRISM 7700 sequence detector system (Applied Biosystems, Weiterstadt, Germany) using the modified method as described earlier [10
]. In brief, the polymerase chain reaction (PCR) master mix contained 5 µl 10x TaqMan buffer A (10x: 100 mM KCl, 100 nM Tris-HCl, 100 mM EDTA, 600 nM passive reference A, pH 8.3), 3.5–5.5 mM MgCl2 (all from Perkin-Elmer, Foster City, CA), 200 µM deoxy-adenosine 5'-triphospahte, -cytidine 5'-triphosphate, -guanosine 5'-triphosphate, and 400 µM -uridine 5'-triphosphate, 100–200 nM forward and reverse primer, 100–200 nM fluorogenic probe (all from Applied Biosystems), 1.25 U AmpliTaq Gold DNA polymerase (Perkin Elmer), and RNase-free water in a total volume of 40 µl. Water control (10 µl), diluted standards, or unknown cDNA template were added to a final volume of 50 µl. The following sequence-specific primer pairs and fluorogenic probes were obtained from Applied Biosystems: ß-actin: 5'-ATT GCC GAC AGG ATG CAG AA-3'; 5'-GCC GAT CCA CAC GGA GTA CT-3'; 5'-FAM-CAA GAT CAT TGC TCC TCC TGA GCG CA-tetramethylrhodamine (TAMRA)-3'; IFN-: 5'-TGA ATG TCC AAC GCA AAG CA-3'; 5'-CCT TGA AAC AGC ATC TGA CTC CTT-3'; 5'-FAM-TGG CGA CAG TTC AGC CAT CAC TTG GA-TAMRA-3'. The same master mix was used for samples and standards to reduce tube-to-tube variation. For standard preparation, cDNA fragments of ß-actin and IFN- were cloned into the pCR2.1 plasmid (Invitrogen, Karlsruhe, Germany). Serial tenfold plasmid dilutions (1x107–1x101 molecules) were used in triplicates to generate standard curves. PCR conditions were 2 min at 50°C, 10 min at 95°C for DNA polymerase activation, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C with a final hold for 2 min at 25°C. The fluorescence signals of each well were collected every 7 s, and threshold cycles were set at 10 SD above the baseline fluorescence. The ß-actin content of test samples was defined as normalization factor. Standardized cytokine mRNA quantities (cytokine copies/104 ß-actin copies) were determined by dividing the interpolation-derived values from the cytokine standard curve by the normalization factor.
Statistics
Cytokine levels determined by ELISA are expressed as mean ± SD of triplicates from a representative experiment out of three using different pairs of PBMC donors unless indicated otherwise. Data were analyzed using GraphPad Prism Version 3.02 (GraphPad Software, San Diego, CA). For mean comparisons, Student’s unpaired two-tailed t-test or one-way ANOVA and an appropriate post-test were used as indicated. P values <0.05 were considered significant.
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production in MMLC secretion of untreated PBMC and allogeneic-activated PBMC (MMLC) over 5 days of incubation (Table 1
). The basal concentration of IFN- in culture supernatants was below 0.1 ng/ml. No increase of IFN- levels could be observed in unstimulated PBMC, whereas the activation in MLC resulted in elevated cytokine levels with maximum levels at days 4 and 5 up to 7 ng/ml. For the activation of cells of the innate immunity, we added 10 ng/ml LPS to PBMC. LPS raised the IFN- release in PBMC continuously over 5 days up to 22 ± 1 ng/ml. The addition of 0.1 ng/ml LPS to MMLC resulted in higher IFN- levels than in PBMC alone activated with 10 ng/ml LPS. This enhanced cytokine secretion was caused solely by LPS, as preincubation of LPS with PmB completely blocked the additional cytokine release (Table 1)
. As the IFN- response to the different stimuli tested here increased continuously until day 5, we selected the suboptimal stimulation until day 4 for further analyses. |
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Table 1. Kinetics of IFN- Production (ng/ml) after LPS Stimulation and Treatment with Polymyxin B (PmB)
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production in a dose-dependent manner. The addition of 0.1, 1, and 10 ng/ml LPS to MMLC resulted in a clear, dose-dependent increase of IFN- release compared with LPS-untreated MMLC (Fig. 1
). Compared with basal levels in three independent MMLC experiments, IFN- levels were only slightly increased by 0.01 ng/ml LPS (1.6-fold), whereas LPS stimuli of 0.1, 1, and 10 ng/ml led to 6.2-, 10.1-, and 10.8-fold elevated IFN- levels (P<0.01 for all three stimulations).
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Figure 1. Dose-dependency of LPS-mediated IFN- release in MMLC on day 4. For MMLC culture, PBMC of two unrelated donors were mixed in equal numbers. Different concentrations of LPS were added at the onset of culture. All cultures were performed in triplicates; supernatants were harvested after 4 days. One representative experiment out of three is shown (analysis with ANOVA and Dunnett’s post-test; **, P<0.01, compared with untreated MLC).
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mRNA amounts revealed an enhanced transcription in the presence of LPS, as IFN- mRNA levels in the LPS-stimulated MMLC were two- to threefold higher than in untreated MMLC (Fig. 2
). This increase of IFN- mRNA and protein levels is not a result of an elevated cell number, as LPS did not enhance the proliferation of cells compared with untreated MMLC as measured by thymidine incorporation after 5 days of incubation (Fig. 3
). Thus, LPS dose-dependently enhances IFN- transcription and protein synthesis without inducing cell proliferation.
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Figure 2. Kinetics of IFN- in untreated and LPS-treated MMLC. LPS (1 ng/ml) was added at the onset of culture. At each time point, cells were harvested for IFN- mRNA analysis (lines). At days 1, 2, 4, and 5, culture supernatant was stored for IFN- determination by ELISA (bars). IFN- protein levels of untreated MMLC (open squares, open bars) and LPS-treated MMLC (circles, shaded bars) are expressed as means ± SD (**, P<0.01; ***, P<0.001; unpaired two-tailed t-test).
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Figure 3. Effect of LPS and allogeneic activation on thymidine incorporation. For MMLC culture, PBMC of two unrelated donors were mixed in equal numbers and compared with PBMC cultures of only one donor. LPS (0.1 ng/ml) was added at the onset of culture. After 5 days of incubation, cultures were pulsed with 1 µCi [3H] thymidine per well and analyzed after 18 h. Data are given as means ± SD of four independent experiments performed in triplicates and were compared by ANOVA with Bonferroni’s multiple comparison post-test (**, P<0.01).
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release release in MMLC, the effect of neutralizing mAb against IL-12 and IL-18 was studied. First, we tested the required amount of mAb in MMLC to reduce IFN- release induced by the corresponding cytokine. Antibody concentrations of 1 µg/ml anti-IL-12 mAb and anti-IL-18 mAb were sufficient to reduce the IL-12-enhanced IFN- production by 70% and the IL-18-enhanced IFN- production by 50% (Fig. 4A
).
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Figure 4. Effect of anti-IL-12 Ab and anti-IL-18 Ab in LPS- and cytokine-treated MMLC. Neutralizing mAb against IL-12 or IL-18 were added at the onset of culture to corresponding recombinanat (r)IL-12- and IL-18-treated (A) or to LPS-treated MMLC (B). Supernatants were harvested after 4 days. Values are expressed as means ± SD of triplicates of one representative experiment out of three. Data were compared by unpaired two-tailed t-test (A) and ANOVA with Bonferroni’s multiple comparison post-test (B; **, P<0.01; ***; P<0.001, compared with mAb-untreated culture).
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production to 57.4 ± 1.4% (P<0.05, n=3, repeated measures ANOVA with Dunnett’s post-test), whereas the addition of anti-IL-18 mAb alone resulted in a decrease to 83.4 ± 1.7% (n=3), which was not statistically significant. The treatment with anti-IL-12 and anti-IL-18 mAb yielded IFN- levels of 51.6 ± 8.8% (P<0.01, compared with LPS-treated MLC) and therefore, did not result in significant, synergistic effects of both antibodies. It is interesting that the same pattern could be detected in MMLC without LPS treatment: In the same set of three experiments, anti-IL-12 mAb and anti-IL-18 mAb reduced IFN- secretion to 55.9 ± 23.7% (P<0.01) and 72.4 ± 23.7% (P<0.05), respectively. In the presence of mAb, the IFN- concentration was 47.6 ± 15.4% (P<0.01). Thus, the IFN- release in low-dose, LPS-treated and in LPS-untreated MMLC seems to depend mainly on IL-12 and to a lesser extent on IL-18.
We further analyzed the effects of recombinant IL-12 and IL-18 on IFN- release in MMLC. The addition of IL-12 and IL-18 to MMLC in different concentrations resulted in a dose-dependent increase of IFN- release up to tenfold compared with untreated MMLC. Whereas only trace amounts of IL-12 induced a strong cytokine release, the doses of IL-18 required for comparable results were 2000-fold higher (Fig . 5A
and 5B
). Adding both cytokines simultaneously to MMLC in nonstimulating concentrations increased the IFN- levels significantly compared with the single cytokines (Fig. 5C)
. This suggests a synergy of both cytokines requiring high amounts of IL-18 and very low concentrations of IL-12.
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Figure 5. Effect of IL-12 and IL-18 on IFN- secretion in MMLC on day 4. rIL-12 (A) or rIL-18 (B) was added individually or in combination (C) at the onset of culture. Supernatants were harvested after 4 days. Values are expressed as means ± SD of triplicates of one representative experiment of three experiments. Data were compared by ANOVA with Dunnett’s post-test (A and B; compared with untreated MMLC) or with Bonferroni’s multiple comparison post-test (C; **, P<0.01; ***, P<0.001).
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production in the presence of LPS release into the supernatant after 4 days. Recombinant IL-4 could almost block LPS-mediated IFN- release when added in physiologically occurring concentrations of 20 ng/ml and below (Fig. 6A
). Additionally, we tested the effect of the highly immunosuppressive steroid hydrocortisone on cytokine release. Hydrocortisone blocked even in low concentrations 85% of IFN- release after LPS activation (Fig. 6B)
. Therefore, the MMLC test system described here is sensitive to very low levels of selective immunomodulatory drugs and general immunosuppressive agents.
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Figure 6. Modification of IFN- secretion by IL-4 and hydrocortisone in MMLC on day 4. IL-4 (A) and hydrocortisone (B) were added simultaneously with LPS at the onset of cultures. Supernatants were harvested after 4 days. IFN- values are expressed as means ± SD of triplicates of one representative experiment out of two. Data were compared with LPS-treated MMLC cultures by ANOVA with Dunnett’s post-test (**, P<0.01).
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secreted by T lymphocytes and natural killer (NK) cells, which have been activated by innate-immune cells, is the predominant cytokine in MLC [12
]. Human leukocyte antigen (HLA) differences are responsible for the induction of IFN- release, but no clear correlation between number of HLA differences and IFN- amount could be shown [13
]. Although the clinical value of this system initially proposed as an in vitro model for the prediction of graft rejection in transplantation remains controversial [14
15
16
], the MLC is nevertheless an important model for alloactivation of T cells upon stimulation by APC primarily belonging to innate immunity. As the MLC represents a complex system containing all hematopoietic cell types of innate and adaptive immunity, it should be possible to adapt this system for other immunological purposes, which require a combination of physiological conditions yielding valid and reliable results and would otherwise need in vivo experiments. Our objective was the development of a MMLC assay that could be used for the characterization of known drugs and the screening for novel, therapeutic agents with immunomodulatory properties that might be of interest for the treatment of inflammatory processes that are associated with atherosclerosis and type 2 diabetes (Fig. 7
).
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Figure 7. Schematic representation of the interaction between innate and adaptive immunity in MMLC. Allogeneic stimulation induces an adaptive T cell response via major histocompatiblity complex/peptide–T cell receptor interaction. The addition of LPS strongly increases the activation state and expression of costimulatory signals by innate-immune cells (APC) and thereby potentiates T cell activation and IFN- secretion. These conditions mimic local or systemic inflammation in vivo and therefore provide an appropriate test for immunomodulatory drugs.
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B and transcription of cytokine-encoding genes [17
, 18
]. Upon activation with LPS, human monocytes and macrophages secrete IL-6, IL-1ß, and the T helper cell type 1 (Th1)-promoting cytokines IL-12 and IL-18 [19
]. In combination with IFN-, these innate immunity-derived cytokines in turn induce T cell proliferation and cytokine secretion. In addition to the induction of Th1-immune mediators, LPS has also been reported to induce Th2 reactivity. In animal models of autoimmune diabetes, LPS can suppress as well as enhance autoimmune diabetes [20
21
22
23
]. Which of the two prevails depends on the cell type treated with LPS (in vitro and in cell transfer studies) and the immunological state of the organism treated with LPS [21
]. In cocultures of nonirradiated PBMC from pairs of unrelated, healthy donors, we found that the expected cytokine release could be elevated dose-dependently by very low LPS levels (100 pg/ml corresponding to 50 pg/106 cells) without enhancing cell proliferation. These data are in contrast to previous observations, which described increased cell proliferation in classical, one-way MLC by LPS doses of only 5 pg/ml corresponding to 20 pg LPS/106 cells [24 , 25 ] but might be explained by differences in medium composition. In our study, the cell-culture medium was supplemented with 5% human serum from a lot that has been selected for low stimulating activity in MMLC assays and might contain agents that increase the threshold dose for LPS-induced proliferation.
The LPS-mediated IFN- release in the MMLC system is mainly mediated by IL-12 and to a lesser extent by IL-18. IL-12 and IL-18 share important functional similarities. IL-12 is considered as the main IFN--inducing cytokine in T cells and promotes Th1 differentiation [26
]. IL-18 acts on Th1 cells and synergizes with IL-12 in T cell activation and IFN- induction but can also activate NK cells independently of IL-12 [27
, 28
]. Contrary to our findings, Kohka and co-workers [29
] reported almost complete blocking of LPS-enhanced IFN- release in MLC by neutralizing mAb against IL-12 or IL-18, suggesting redundant effects of both cytokines. However, their MLC was based on high-dose LPS activation (5 µg/ml), which represents a nonbiological concentration of LPS. Such an extreme stimulus might result in different mechanisms of IFN- induction compared with very low-dose LPS stimulation protocols [29
].
It is conceivable that additional immune mediators besides IL-12 and IL-18 trigger optimal IFN- release upon allogeneic and/or LPS stimulation in MMLC. It is interesting that LPS has been demonstrated to stimulate regulatory T cells independently of interaction with innate-immune cells [30
], and LPS-activated monocytes also produce IL-15 and other cytokines that can contribute to optimal IFN- release by NK cells in synergy with IL-12 [31
]. Therefore, the MMLC system as described here reflects the complexity of the physiological in vivo situation and should allow for the identification of immunomodulatory substances. The sensitivity of the test system is exemplified by addition of different doses of the Th2 cytokine IL-4 and hydrocortisone. As described previously [32
], IL-4 counteracts IFN- release, which might be a result of effects on different cell types. IL-4 acts on monocytes by decreasing CD14 expression and therefore impairing LPS binding [33
] and by inhibiting LPS-induced expression of IL-12 p40, leading to considerably lower IL-12 p70 and p40 protein release [34
]. Furthermore, IL-4 decreases the expression of the high-affinity IL-12 receptor on T cells [35
]. Similar to low-dose IL-4 treatment, a concentration of only 1 µg/ml hydrocortisone sufficed to strongly inhibit IFN- production in LPS-activated MMLC.
In the strict sense, the LPS-activated MMLC mimics the situation of transplant patients having received standard conditioning regimens including total body irradiation. The combination of conditioning-related toxicity and the release of inflammatory cytokines such as IL-12 during graft-versus-host disease (GVHD) leads to damage of the gastrointestinal tract and subsequent leakage of LPS into the circulation [36 , 37 ]. Analysis of a murine GVHD model demonstrated that LPS accumulated in liver and spleen and also appeared in serum at concentrations of up to 6 ng/ml [38 ].
Taken together, our findings elucidate the mechanisms in MMLC of allogeneic activation and stimulation by very low LPS concentrations of 0.1 and 1 ng/ml, which represent a physiological stimulus. We found that IL-12 and to a lesser extent IL-18 are important mediators of activation, which are presumably released by cells of innate immunity. This leads to subsequent activation of adaptive immunity, which can be measured as IFN- release. Immunomodulatory agents such as IL-4 and hydrocortisone can interfere at low concentrations with the interplay of innate and adaptive immunity, leading to an altered IFN- response. Therefore, the MMLC system offers the required complexity of an immunological test system in combination with low expense in terms of financial resources and time.
Received October 1, 2003; revised December 4, 2003; accepted December 8, 2003.
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inhibits human T lymphocyte and natural killer cell activation J. Immunol. 147,1445-1452[Abstract]-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon- production Eur. J. Immunol. 26,1647-1651[Medline] production by IL-12 and IL-18 Curr. Opin. Immunol. 10,259-264[CrossRef][Medline] by natural killer cells in vitro J. Clin. Invest. 96,2578-2582 and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression Eur. J. Immunol. 27,647-652[Medline]
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