Dimerization of major histocompatibility complex class I on the surface of THP-1 cells stimulates the expression of inducible nitric oxide synthase and subsequent nitric oxide release
Summary
We show that dimerization of major histocompatibility complex (MHC) class I on a human monocytic cell line, THP-1, induces nitric oxide (NO) synthesis. Cells cultured in the presence of a human MHC class I-specific monoclonal antibody produced significant amounts of NO after 72 hr. Reverse transcription–polymerase chain reaction and flow cytometry analysis revealed that the cells synthesized detectable levels of inducible NO synthase mRNA and protein. These effects were not seen after treatment with monovalent Fab fragments or Fc fragments of the same antibody, or after treatment with a control antibody. These data show a link between innate and acquired immune mechanisms mediated by NO and MHC class I.
Abbreviations
-
- NO
-
- nitric oxide
-
- iNOS
-
- inducible nitric oxide synthase
Introduction
The link between natural and specific immunity has recently been the focus of attention. 1 In most cases when specific immune mechanisms are activated, natural mechanisms are also enhanced. One example is the possible relationship between ligation of cell surface receptors of monocytic cells and release of nitric oxide (NO). We have focused on major histocompatibility complex (MHC) class I as a central group of receptors that are involved in antigen presentation.
NO is produced by the immune system in response to many threats, including bacterial, viral and fungal infections and tumours. 2–4 Immune cells, such as macrophages, monocytes or dendritic cells, use the enzyme inducible nitric oxide synthase (iNOS or NOS II) to produce NO from l-arginine in response to stimulants such as lipopolysaccharide (LPS) 5 or interferon-γ (IFN-γ).6, 7 NO responses in vitro and in vivo are very well characterized in murine macrophages,5, 8 but not so in human cells. Several in vitro studies have failed to induce NO synthesis in human cells after stimulation with LPS and IFN-γ,9,10 whilst other studies have succeeded. 11 Despite this inconsistency, many factors have been reported to promote NO release by human immune cells, among them LPS, 12 IFN-γ13 interleukin-4 (IL-4), 14 CD23 ligation15, 16 and tumour cell co-culture. 9 It has been suggested that cross-linking of CD69 on the surface of human peripheral blood monocytes induces NO synthesis. 10 CD69 appears to be involved in tumoricidal activity and this suggests that CD69 cross-linking may be an important part of monocyte activation.
The role of MHC as an intermediate in cell signalling is unclear at present but it has been shown that anti-MHC antibodies inhibit NO release by human peripheral blood monocytes co-cultured with colorectal carcinoma cells. 17 It has also been reported that cross-linking of MHC class II antigens by monoclonal antibodies induces NO production by murine monocytes; 18·19 However, the role of MHC class I in NO up-regulation has not been investigated so far. It is known, however, that ligation and cross-linking of MHC class I can lead to signal transduction in T cells 20 and can induce proliferation of lymphocytes. 21 Thus we investigated the role of MHC class I cross-linking in a human monocytic leukaemic cell line, THP-1, to see whether there is a link between antigen presentation and NO synthesis, two important parts of the adaptive immune response.
Materials and methods
Cell culture
THP-1 cells (human monocytic leukaemic cell line) were obtained from the European Collection of Animal Cell Cultures (ECACC) Salisbury, UK and cultured in macrophage serum-free medium (MSFM) (GIBCO-BRL, Paisley, UK) in filter-cap flasks. The cultures were maintained at between 0·5 × 106 and 1 × 106 cells/ml at 37° in a 5% CO2 atmosphere. All experimental and control cultures were initially seeded at a concentration of 1 × 106 cells/ml.
W6/32 Fab fragment, Fc fragment and F(ab′)2 preparation
W6/32 antibody [human MHC class I-specific; immunoglobulin G2 (IgG2) isotype]22 was purified on a HiTrap Protein A column (Amersham Pharmacia, Buckinghamshire, UK). Fab and Fc fragments were generated by digestion with papain and then separated on a HiTrap Protein A column. F(ab′)2 was generated by pepsin digestion and also purified on a HiTrap Protein A column. The concentrations of the IgG, Fab, Fc and F(ab′)2 were calculated by measuring the absorbance at 280 nm. The Fab and Fc fractions were further purified by size exclusion high-performance liquid chromatography (HPLC) to remove undigested IgG. The IgG, Fab, Fc and F(ab′)2 fractions were sterilized by sterile syringe filtration with a 0·2-µm-pore size Acrodisc filter (Sartorius, Göttingen, Germany).
Confocal microscopy
Expression of iNOS protein was examined using rabbit anti-human iNOS polyclonal antibody (Biomol, Exeter, UK) as the primary antibody and swine anti-rabbit fluorescein isothiocyanate (FITC) secondary antibody. The cells were first fixed with 4% paraformaldehyde and permeabilized with 0·1% Triton X-100. The cells were analysed on a Bio-Rad confocal microscope (Bio-Rad, Hertfordshire, UK) at × 60 magnification.
Flow cytometry
Cellular levels of iNOS were assessed by single-colour flow cytometry. The cells were labelled as for confocal microscopy and analysed on a Becton Dickinson FACScan (Oxford, UK). The data was analysed with lysis ii software.
Nitrite detection
The nitrite present in the culture medium was determined by a modified Griess reaction. 23 The culture medium was centrifuged at 12 000 g for 5 min in a bench-top centrifuge. The supernatant was then filtered through a 0·2-µm-pore size syringe filter to remove cell debris. Then, 100 µl of supernatant was mixed with 50 µl 1% sulphanilimide in 5% phosphoric acid and 50 µl 0·1% naphthylene ethylene diamine in a 96-well plate and incubated at 4° for 10 min. The absorbance was assessed using a multi-well plate reader at 540 nm. A sodium nitrite standard curve was used to determine actual nitrite concentrations.
Reverse transcription–polymerase chain reaction (RT-PCR)
For mRNA isolation, 0·5 × 106 cells were washed in phosphate-buffered saline (PBS) and the mRNA was isolated by binding to Hybond mAP (messenger affinity paper) (Amersham, Bucks., UK). 24 Reverse trancription using Moloney murine leukaemia virus (M-MLV) reverse transcriptase was carried out with an oligo-dT17 primer and the cDNA was then amplified using PCR. The gene-specific primer sequences were as given in Table 1. The iNOS cDNA was further amplified with a second round of PCR using the same programme and primers, but using 3 µl of PCR product from the first round as the template.
| Primer sequence | PCR | Programme | Fragment | ||
|---|---|---|---|---|---|
| iNOS | 5′-TCCGAGGCAAACAGCACATTCA-3′ | 92° | 1 min | ||
| 5′-GGGTTGGGGGTGTGGTGATGT-3′ | 46° | 1 min 30 seconds | 35 cycles | 446 bp | |
| 72° | 1min 30s | ||||
| β-Actin | 5′-AGCGGGAAATCGTGCGTG-3′ | 92° | 1 min | ||
| 5′-CAGGGTACATGGTGGTGCC-3′ | 60° | 1 min 30 seconds | 35 cycles | 300 bp | |
| 72° | 1 min 30 seconds |
- bp, base pairs.
The PCR reaction was carried out using Blue-Taq MegaMix (Helena Biosciences, Sunderland, UK). The mixture contained 3 µl cDNA, 1·6 µm sense and antisense primer and 1 U Taq polymerase in a total reaction mixture of 25 µl. The PCR was carried out on a Biometra thermal cycler (Göttingen, Germany). Electrophoresis of the PCR products was carried out on a 2% agarose gel containing 1 µg/ml ethidium bromide. The DNA ladder was 80–1000 base pairs.
Results
NO production following MHC class I dimerization
One indicator of iNOS stimulation is the presence of nitrites and nitrates in the cell culture supernatant. These accumulate in varying proportions depending on many factors, but nitrite concentration acts as a good guide to the amount of NO produced and was measured after 72 hr to allow the nitrite to accumulate. The time of culture was chosen after preliminary experiments showed significant nitrite was detectable after 72 hr (unpublished observations). The culture supernatant was assayed by the Griess reaction for nitrite levels as shown in Fig. 1. The mean nitrite levels for the treatments were subjected to a two-tailed unpaired t-test. The cells stimulated by intact W6/32 or W6/32 F(ab′)2 produced significant levels of nitrite compared to non-stimulated cells. Cells that were stimulated with control IgG, W6/32 Fab fragments or W6/32 Fc fragments did not produce significantly different nitrite levels from the non-stimulated cells. This suggests that only cells stimulated by MHC class I dimerization produced NO.
Cell culture supernatant nitrite levels of THP-1 cells. The error bars represent one SD. Data are presented for resting THP-1 cells (a) and also cells after 72 hr stimulation with 1 μg/ml W6/32 IgG (b), control mouse IgG (c), W6/32 Fab (d), W6/32 Fc (e) and W6/32 F(ab′)2 (f).
iNOS expression following MHC class I dimerization
To verify further that accumulated culture nitrite was due to iNOS activity and not to some other effect, iNOS expression by treated THP-1 cells was analysed by single-colour flow cytometry ( Fig. 2) and fluorescence confocal microscopy ( Fig. 3), using a polyclonal rabbit antibody against the iNOS enzyme. Stimulation of THP-1 cells for 18 hr with control immunoglobulin, Fab fragments or Fc fragments produced no induction of iNOS expression, which is a non-constitutive enzyme. However, treatment of the cells for 18 hr with whole W6/32 IgG or F(ab′)2 fragments induced a low level of iNOS expression. Preliminary experiments (unpublished observations) showed that 18 hr was sufficient time for significant iNOS mRNA and enzyme to be expressed.
Flow cytometric histograms showing the mean levels of iNOS of THP-1 cells after 72 hr culture: (a) no stimulation, (b) whole W6/32 IgG, (c) control mouse IgG, (d) W6/32 Fab, (e) W6/32 Fc and (f) W6/32 F(ab′)2. The upper histograms indicate cells labelled with the secondary antibody only. The lower histograms indicate cells labelled with anti-iNOS and secondary FITC-conjugated antibody.
Confocal microscopy images showing cells labelled with rabbit anti-iNOS and swine anti-rabbit FITC-conjugated secondary antibody (right-hand images). Cells incubated with secondary antibody only are also presented (left-hand images). All images represent an area 93 μm by 62 μm. (a/A) No stimulation; (b/B) whole W6/32 IgG; (c/C) control mouse IgG; (d/D) W6/32 Fab; and (e/E) W6/32 Fc.
The effects of the treatments were investigated further by analysing cell lysates for iNOS mRNA by RT-PCR. The results are shown in Fig. 4. It can be clearly seen in Fig. 3 that iNOS mRNA is present in cells stimulated for 18 hr with whole W6/32 and not in non-stimulated cells or in cells stimulated for 18 hr with control IgG, Fab fragments, or Fc fragments.
A 2% agarose gel showing the results from RT-PCR with cDNA derived from THP-1 cells after 18 hr culture: lane a, no stimulation; lane b, whole W6/32 IgG; lane c, control mouse IgG; lane d, W6/32 Fab; lane e, W6/32 Fc. The top row shows cDNA amplified with primers specific for iNOS, the lower row shows cDNA amplified with primers specific for β-actin.
Discussion
NO is involved in several immunological responses including cellular toxicity,3,25 autoimmunity26,27 and T helper type 1 (Th1) cell development. 28 We show here that MHC class I signal transduction in a human monocytic/macrophage model is interlinked with NO synthesis. MHC class I is expressed on all nucleated cells and has been shown to be a signal-transducing molecule inducing proliferation in human lymphocytes. 21 We demonstrate here that activation of the NO synthesis pathway can also occur following dimerization of MHC class I.
Using the human monocytic leukaemic cell line THP-1, which has some characteristics of human alveolar macrophage 29 cells, we have observed that the NO synthesis pathway is activated after culture with an intact monoclonal IgG or F(ab′)2 directed against MHC class I.
Induction of NO was determined by examining iNOS mRNA (by RT-PCR), iNOS enzyme expression (by flow cytometry) and the accumulation of NO reaction products (nitrite level by the Griess reaction).
Treatment with monovalent Fab fragments does not cause NO release, iNOS expression, or mRNA expression, so simple ligation or binding of the surface MHC class I molecule is insufficient to stimulate NO synthesis. The induction of the NO pathway cannot be due to ligation of the cell surface Fc receptor because treatment of the cells with W6/32 Fc fragments or control IgG did not elicit NO release or iNOS expression whilst treatment with W6/32 F(ab′)2 fragments did induce NO synthesis.
This then suggests that it is the dimerization of MHC class I molecules on the cell surface that is responsible for the iNOS up-regulation.
Cross-linking of both MHC class I and MHC class II molecules has been shown to lead to signal transduction. Induction of the iNOS pathway in murine macrophages has been shown to follow MHC class II cross-linking.18,19 Cross-linking of MHC class I can induce proliferation in human peripheral blood mononuclear cells 21 and has been shown to induce tyrosine phosphorylation in human T cells. 30
The mechanisms that lead to up-regulation of iNOS following the dimerization of MHC class I are as yet unknown, as is the immunological significance of this pathway. However, the fact that NO production is linked to endogenous antigen presentation by human monocytes suggests that NO may have other roles than those already shown.
In conclusion, we show here that there is a link between innate and acquired immune mechanisms mediated by NO and MHC class I.
