TY - JOUR
AU - Yoshida, R
AB - Summary Mouse monocyte/macrophage major histocompatibility complex (MHC) receptor 1 (MMR1; or MMR2) specific for H-2Dd (or H-2Kd) molecules is expressed on monocytes from non-H-2Dd (or non-H-2Kd), but not those from H-2Dd (or H-2Kd), inbred mice. The MMR1 and/or MMR2 is essential for the rejection of H-2Dd- and/or H-2Kd-transgenic mouse skin onto C57BL/6 (H-2Db Kb) mice. Recently, we found that human leucocyte antigen (HLA)-B44 was the sole ligand of human MMR1 using microbeads that had been conjugated with 80 types of HLA class I molecules covering 94·2% (or 99·4%) and 92·4% (or 96·2%) of HLA-A and B molecules of Native Americans (or Japanese), respectively. In the present study, we also explored the ligand specificity of human MMR2 using microbeads. Microbeads coated with HLA-A32, HLA-B13 or HLA-B62 antigens bound specifically to human embryonic kidney (HEK)293T or EL-4 cells expressing human MMR2 and to the solubilized MMR2-green fluorescent protein (GFP) fusion protein; and MMR2+ monocytes from a volunteer bound HLA-B62 molecules with a Kd of 8·7 × 10−9 M, implying a three times down-regulation of MMR2 expression by the ligand expression. H-2Kd (or H-2Dd) transgene into C57BL/6 mice down-regulated not only MMR2 (or MMR1) but also MMR1 (or MMR2) expression, leading to further down-regulation of MMR expression. In fact, monocytes from two (i.e. MMR1+/MMR2+ and MMR1–/MMR2–) volunteers bound seven to nine types of microbeads among 80, indicating ≤ 10 types of MMR expression on monocytes. The physiological role of constitutive MMRs on monocytes possibly towards allogeneic (e.g. fetal) cells in the blood appears to be distinct from that of inducible MMRs on macrophages toward allografts in tissue. down-regulation, HLA, humam, monocyte, receptor Introduction Immunity in most of the studied vertebrates is dependent upon two integrated immune systems, the innate and the adaptive, whose principal function is to protect the host against viral or bacterial infection [1–4]. In the innate immunity, which includes the action of Toll-like receptors (TLRs) towards microbial pathogens, TLRs recognize pathogen-associated molecular patterns in microbial pathogens [5–8]. In the case of allogeneic non-self, however, antigens recognized as non-self are histocompatibility antigen-2 (H-2) molecules in mice and human leucocyte antigen (HLA)-A, B and C molecules in humans, which are very similar to each other with sometimes ≥ 97% amino acid identity [9]. In addition, using skin grafts from tetraparental (allophenic) donors, Minz and Silvers [10,11] showed that the rejection process that destroyed the allogeneic cells left the syngeneic cells intact, suggesting specific cytotoxicity of recipient cells against allogeneic, but not syngeneic, cells. Thus, the receptor ligand mechanism of allorecognition appears to be distinct from that of non-self recognition by pattern recognition receptors. Moreover, athymic nude rodents fail to reject allografts [12], and therefore it has been assumed that T lymphocytes are the effector cells for allograft rejection. In the case of adaptive immunity, however, how the immune system distinguishes self-tissues from allogeneic tissues has been controversial. On one hand, during the 1970s and later, several groups reported that the T cell-deficient nude mouse failed to reject skin or organ allografts, that allograft rejection was restored by adoptive transfer of T cells to the nude mouse, and that cytotoxic T lymphocytes (CTLs) were cytotoxic against donor-type lymphoblasts [13–16]. Therefore, it has been recognized that the rejection of allografts expressing non-self major histocompatibility complex (MHC) is mediated by CTLs. On the other hand, in the period 1992–96, several groups using β2m [17], CD8 [18] or CD4 [19] knock-out mice reported that neither CTLs nor natural killer cells were essential for the rejection of skin or organ allografts and that non-cytotoxic T helper type 1 (Th1) cells were absolutely required for initiating allorejection. Recently, several mouse or human studies have implicated monocytes or macrophages as playing a more immediate role in allograft rejection than suspected previously [20–22]. In 1988, we transplanted intraperitoneally (i.p.) Meth A tumour cells, a CTL-resistant cell line [23] of BALB/c origin, into C57BL/6 mice and found, unexpectedly, that such cells (allografts) were rejected [24]. In 1991, among various types of cells infiltrating into the rejection site, allograft-induced macrophages (AIM) were found to be the major population of effector cells responsible for this rejection [25]. These AIM were also induced as the major effector cells in the rejection site after BALB/c skin transplantation onto C57BL/6 mice [26]. Of particular interest, AIM (H-2b) exhibited H-2d haplotype-specific cytotoxic activity against allografts (H-2d; e.g. BALB/c skin components and Meth A tumour cells) in a cell-to-cell contact-dependent, but Fas-, perforin-, cytophilic antibody- and soluble factor [e.g. tumour necrosis factor (TNF)-α, nitric oxide (NO)]-independent manner [27–38]. In 2006, we isolated two cDNA clones encoding novel MHC receptors on AIM for allogeneic MHCs (H-2Dd and H-2Kd) by using anti-AIM monoclonal antibodies (mAbs; R15 and R12) and H-2Dd and H-2Kd tetramers and named them macrophage MHC receptor 1 (MMR1) and MMR2 [39,40]. Although there is a more than 94% amino-acid homology between α1 and α2 chains of various H-2 class I molecules such as H-2Dd, H-2Db, H-2Dk, H-2Kd, H-2Kb, H-2Kk and H-2Ld [9], H-2Dd (or H-2Kd), but not H-2Db, H-2Dk, H-2Kd (or H-2Dd), H-2Kb, H-2Kk and H-2Ld, tetramers bind specifically to MMR1 (or MMR2). The binding of H-2Dd (or H-2Kd) molecules to MMR1 (or MMR2) with a Kd of 2∼3 × 10−9 M was suppressed by the addition of anti-H-2Dd (or anti-H-2Kd), but not that of anti-H-2Kd (or anti-H-2Dd), antibody, suggesting direct recognition of allo-MHC class I molecules by MMRs on AIM. Of particular interest, mouse MMR1 (or MMR2) specific for H-2Dd (or H-2Kd) molecules was expressed on monocytes from non-H-2Dd (or non-H-2Kd; i.e. non-ligand), but not those from H-2Dd (or H-2Kd; i.e. ligand), inbred mice [41,42]; and MMR1–/– and/or MMR2–/– C57BL/6 mice failed to reject H-2Dd- and/or H-2Kd-transgenic mouse skin grafts onto C57BL/6 mice [43,44]. Moreover, we isolated cDNAs encoding the human homologues of mouse MMR1 and MMR2 and found their expression on at least some of the peripheral blood mononuclear cells (PBMCs) or monocytes, but not on granulocytes or lymphocytes [41,42]. The human MMR1 on monocytes is a novel receptor specific for HLA-B44 among 80 types of HLA class I molecules [41] covering 94·2% (or 99·4%) and 92·4% (or 96·2%) of the HLA-A and B molecules, respectively, of Native Americans (or Japanese) [45,46], although HLA-B44 has ≥ 94% amino-acid identity to HLA-B47, B13, B53, B49, B37, B40, B45, B27 and B57 molecules [9]. In the present study, we explored the ligand specificity of human MMR2 using microbeads and found HLA-A32, HLA-B13 and HLA-B62 to be specific ligands. We also showed, by using the microbeads and monocytes from two volunteers, one expressing both MMR1 and MMR2 and the other expressing neither, that only ≤ 10 types of MMRs were expressed on their monocytes after down-regulation by the ligand expression. Materials and methods Animals Specific pathogen-free C57BL/6 mice (7 weeks of age) were purchased from Japan SLC (Hamamatsu, Japan). Transgenic mice carrying the H-2Dd or H-2Kd gene were generated on a C57BL/6 background, as described previously [43]. H-2DdKd double-transgenic mice were obtained by breeding H-2Dd-transgenic mice (Accession no. CDB0468T) with H-2Kd-transgenic ones (Accession no. CDB0467T: http://www.cdb.riken.jp/arg/TG%20mutant%20mice%20list.html). The animals were housed in our animal facility under specific pathogen-free conditions in an air-conditioned room at 23 ± 2°C and ≈50% humidity. The experiments were carried out in accordance with the Guidelines on Animal Experiments of Osaka Medical College and the Japanese Government Notification on Feeding and Safekeeping of Animals (Notification no. 6 of the Prime Minister's Office). The experimental protocol was approved by the Review Committee for Animal Experiments of Osaka Medical College. Reagents Phycoerythrin (PE)-labelled H-2Dd, H-2Kd and HLA-B62 (or HLA-B1501) pentamers were purchased from ProImmune (Oxford, UK). The peptide sequences for the H-2Dd, H-2Kd and HLA-B62 pentamers were RGPGRAFVTI, SYIGSINNI and VQKDDIQIRF (or RLRPGGKKKY), respectively. Homology search A search for sequence homology was conducted using blast (http://blast.genome.jp/), and the sequence alignment was performed by use of the clustal w Multiple Sequence Alignment Program (http://align.genome.jp/). Preparation of PBMCs and monocytes Human or mouse peripheral blood was diluted with an equal volume of phosphate-buffered saline (PBS) containing 5 mM ethylenediamine tetraacetic acid (EDTA), and red blood cells and most of the granulocytes were removed by gradient centrifugation (400 g for 30 min at 20°C) in PBS/Lympholyte®-H or Lympholyte®-M (Cedarlane, Ontario, Canada). With the gate set in the forward-scattering–side-scattering mode, monocytes were sorted using a fluorescence-activated cell sorter (FACS; FACSAria, Becton Dickinson, Mountain View, CA, USA). Cell number and viability Cell number was determined by counting cells in Turk's solution with a haemocytometer. The viability of cells was assessed by the trypan blue exclusion method. Transfection of human embryonic kidney (HEK)293T or EL-4 cells with human MMR2 cDNA The full-length cDNA of human MMR2 was inserted into the pEGFPNI vector, and HEK293T cells were transfected with the linearized plasmid using LipofectamineTM 2000 (Invitrogen, CH Groningen, the Netherlands). Stable transfectants were selected for their resistance to 1 mg/ml of G418 [47]. In the case of EL-4 cells, the full-length cDNA of human MMR2 inserted into a pCAGGS vector was used for transfection of the cells, carried out using the Nucleofector program A-017 (Nucleofector 2; Amaxa, Austin, TX, USA), and stable transfectants were selected for their resistance to G418 (500 μg/ml). Binding of microbeads conjugated with various HLA class I molecules to human MMR2 on HEK293T cells The FlowPRA® Single Antigen Antibody Detection Test (One Lambda, Inc., Canoga Park, CA, USA) is composed of microbeads, each type coated with each of 80 types of purified HLA class I molecules: group 1 beads (HLA-A1, A2, A3, B49, A25, A29, A30 and A26), group 2 beads (A68, A11, A34, A24, A32, A33, A31 and A23), group 3 beads (B51, B13, B18, B35, B62, B45, B60 and B44), group 4 beads (B38, B57, B7, B52, B27, B8, B65 and B55), group 5 beads (B37, B39, B41, B42, B46, B47, B48 and B50), group 6 beads (B53, B54, B56, B58, B59, B61, B63 and B64), group 7 beads (B67, B81, B72, B73, B75, B76, B77 and B78), group 8 beads (A36, A66, A43, A74, A80, B71, B8201 and A69), group 9 beads [Cw1, Cw2, Cw10 (0302), Cw9, Cw4, Cw5, Cw6 and Cw7] and group 10 beads [Cw8, Cw12, Cw14, Cw15, Cw16, Cw17, Cw18 and Cw10 (0304)]. These HLA molecules cover 94·2 (or 99·4)% and 92·4 (or 96·2)% of HLA-A and B molecules of Native Americans (or Japanese), respectively [45,46]. HEK293T cells expressing the enhanced green fluorescent protein (EGFP)-tagged human MMR2 protein were incubated for 30 min in the presence or absence (negative control) of the HLA-coated microbeads. The unbound beads were washed twice with 2 ml of PBS. The washed beads were then suspended in PBS containing 0·5% formaldehyde and analysed by FACS. The binding index was calculated by subtracting the mean fluorescence intensity (MFI) of the negative control from that of HEK293T cells expressing human MMR2. Binding of solubilized MMR2–GFP fusion protein to microbeads conjugated with various HLA class I molecules One ml of precooled lysis buffer was added to the pellet of EL-4 cells (1 × 107 cells). After having been well mixed, the cell lysate was stood on ice for 30 min and then sedimented by centrifugation at 10 000 g for 10 min at 4°C. The supernatant was transferred to a fresh 1·5-ml tube, and EGFP-tagged human MMR2 protein was concentrated and purified using Amicon Ultra-0·5 ml Centrifugal Filters (Merck KGaA, Darmstadt, Germany). The EGFP-tagged human MMR2 protein was incubated for 30 min in the presence or absence (negative control) of each group of the above-mentioned HLA protein-conjugated microbeads. The unbound beads were washed twice with 2 ml of PBS and the washed beads were then suspended in PBS containing 0·5% formaldehyde and analysed by FACS. The binding index was calculated by subtracting the MFI of the negative control from that of the EGFP-tagged human MMR2 protein. Sorting of microbeads coated with HLA-A32, HLA-B13, HLA-B60 or HLA-B62 molecules In the case of HEK293T cells expressing EGFP-tagged human MMR2 protein, the binding index was calculated by subtracting the MFI of the negative control from that of HEK293T cells expressing the EGFP-tagged human MMR2. Because the FlowPRA® Single Antigen Antibody Detection Test is composed of phycoerythrin (PE)-labelled microbeads coated with 80 types of purified HLA class I molecules, and because HEK293T cells had spontaneous red fluorescence, we sorted PE-labelled microbeads coated with HLA-A32, HLA-B13, HLA-B60 or HLA-B62 molecules and then used them to determine the binding index. EL-4 cells expressing human MMR2 protein were incubated for 30 min in the presence or absence (as a negative control) of microbeads conjugated with HLA-A32, HLA-B13, HLA-B60 (as a control) or HLA-B62 proteins. The unbound beads were washed twice with 2 ml of PBS. The washed beads were then suspended in PBS containing 0·5% formaldehyde and analysed by FACS. Skin transplantation The transplantation of full-thickness dorsal skin was performed according to the method of Billingham and Medawar [48], with slight modifications [43]. Reverse transcription–polymerase chain reaction (RT–PCR) Total RNAs from mouse PBMCs were reverse-transcribed to synthesize the first-strand cDNAs, as described previously [39,40]. Primer sets for mouse MMR2 (GenBank Accession no. AB247936; forward: 5′-GGC TAG CAT GTC ACC TAG CCA GAG TGG ACT GTT GG-3′; reverse: 5′-TTG TCG ACC TAT GCT ACC TGG TAA ACA CTG TGA CA-3′; Life Technologies, Carlsbad, CA, USA) and MMR1 (GenBank Accession no. AB206122; forward: 5′-GCC CAT TTG GAT GAA ATG CC-3′; reverse: 5′-GGG TCT CAG TGG CCA CGT CT-3′; Life Technologies) were used to amplify 2049 base pairs (bp) and 1181 bp fragments, respectively, by 35 cycles of RT–PCR. A primer set of mouse β-actin (forward: 5′-TGT GAT GGT GGG AAT GGG TCA G-3′; reverse: 5′-TTT GAT GTC ACG CAC GAT TTC C-3′; Kurabo, Osaka, Japan) was used to amplify a 514-bp fragment by 30 cycles of RT–PCR. The PCR was conducted in a GeneAmp PCR System apparatus (9700; PE Applied Biosystems, Foster City, CA, USA). The PCR products were electrophoresed on 2% agarose gels and analysed after ethidium bromide staining. Binding of microbeads conjugated with various HLA class I molecules to human PBMCs PBMCs were incubated with fluorescein isothiocyanate-labelled mouse anti-human CD14 mAb (Dako, Glostrup, Denmark) in the presence of control mouse serum on ice for 15 min. Fluorescein-labelled PBMCs or monocytes were incubated at 20°C for 30 min with HLA (+) or HLA (–) microbeads. The binding index was calculated by subtracting the MFI of the negative control [HLA (–)] from that of the test [HLA (+)] samples. Results Specific binding of HLA-A32-, HLA-B13- or HLA-B62-conjugated microbeads to HEK293T or EL-4 cells expressing human MMR2 To examine which HLA class I molecule(s) was able to bind to human MMR2, we performed a binding assay using 80 types of HLA-conjugated microbeads. Human MMR2 cDNA (≈2·4-kb) tagged with EGFP was stably expressed in HEK293T cells. The binding of HLA-conjugated microbeads to human MMR2-transfected HEK293T cells could be detected as MFI of GFP+ cells by FACS. Flow cytometric analysis showed that microbeads conjugated with HLA-A32, HLA-B13 or HLA-B62 bound significantly to the HEK293T cells expressing GFP-tagged human MMR2 protein (Fig. 1a). In the case of HLA-B51- and HLA-B47-coated microbeads, the binding to the MMR2+ HEK293T cells was less significant, and the 75 types of other HLA class I molecule-coated beads showed no binding to the MMR2+ HEK293T cells. Fig. 1 Open in new tabDownload slide Binding of beads conjugated with various human leucocyte antigen (HLA) class I proteins to human monocyte/macrophage major histocompatibility complex (MHC) receptor 2/enhanced green fluorescent protein (MMR2/EGFP)-transfected human embryonic kidney (HEK)293T cells. (a) Cellular monocyte/macrophage MHC receptor (MMR)2 detected as mean fluorescence intensity (MFI) of green fluorescence protein (GFP)+ cells by fluorescence-activated cell sorter (FACS). The MFI fold = (MFI of HLA+-beads bound to human MMR2+-HEK293T cells – MFI of HLA+-beads alone)/(MFI of HLA–-beads bound to human MMR2+-HEK293T cells – MFI of HLA–-beads alone). Ten groups of beads conjugated with various HLA proteins were incubated with or without (as a negative control) HEK293T transfectants. Each value represents the mean ± SD of 3 different experiments (n = 9). The difference in binding between sample and control (HLA-B60: a control in Table 1) is significant (*P < 0·02; **P < 0·005; ***P < 0·002) according to Student's t-test. (b) Soluble MMR2 protein detected as MFI of MMR2-GFP fusion protein by FACS. The MFI fold = (MFI of HLA+-beads bound to human MMR2-GFP fusion protein – MFI of HLA+-beads alone)/(MFI of HLA–-beads bound to human MMR2-GFP fusion protein – MFI of HLA–beads alone). Ten groups of beads conjugated with various HLA proteins were incubated with or without (as a negative control) MMR2-GFP fusion protein. Each value represents the mean of two different experiments (n = 2) for group 3 and the MFI fold of a single experiment (n = 1) for nine other groups. Fig. 1 Open in new tabDownload slide Binding of beads conjugated with various human leucocyte antigen (HLA) class I proteins to human monocyte/macrophage major histocompatibility complex (MHC) receptor 2/enhanced green fluorescent protein (MMR2/EGFP)-transfected human embryonic kidney (HEK)293T cells. (a) Cellular monocyte/macrophage MHC receptor (MMR)2 detected as mean fluorescence intensity (MFI) of green fluorescence protein (GFP)+ cells by fluorescence-activated cell sorter (FACS). The MFI fold = (MFI of HLA+-beads bound to human MMR2+-HEK293T cells – MFI of HLA+-beads alone)/(MFI of HLA–-beads bound to human MMR2+-HEK293T cells – MFI of HLA–-beads alone). Ten groups of beads conjugated with various HLA proteins were incubated with or without (as a negative control) HEK293T transfectants. Each value represents the mean ± SD of 3 different experiments (n = 9). The difference in binding between sample and control (HLA-B60: a control in Table 1) is significant (*P < 0·02; **P < 0·005; ***P < 0·002) according to Student's t-test. (b) Soluble MMR2 protein detected as MFI of MMR2-GFP fusion protein by FACS. The MFI fold = (MFI of HLA+-beads bound to human MMR2-GFP fusion protein – MFI of HLA+-beads alone)/(MFI of HLA–-beads bound to human MMR2-GFP fusion protein – MFI of HLA–beads alone). Ten groups of beads conjugated with various HLA proteins were incubated with or without (as a negative control) MMR2-GFP fusion protein. Each value represents the mean of two different experiments (n = 2) for group 3 and the MFI fold of a single experiment (n = 1) for nine other groups. To confirm the results of the binding assay using 80 types of HLA-conjugated microbeads, we incubated MMR2–GFP fusion protein with the HLA-conjugated microbeads (Fig. 1b). PE-labelled microbeads conjugated with HLA-B13 (MFI fold = 22·8), HLA-B62 (MFI fold = 21·9) and HLA-A32 (MFI fold = 7·9) bound to the MMR2-GFP fusion protein, in that order. In contrast, HLA-B51- and HLA-B47-coated microbeads did not bind to the MMR2–GFP fusion protein, and the other 75 types of HLA class I-coated microbeads were inactive towards the fusion protein. To confirm the results of the binding assay using the 80 types of HLA-conjugated microbeads with another cell type, we sorted PE-labelled microbeads coated with HLA-A32, HLA-B13, HLA-B60 (as a negative control) or HLA-B62 molecules and incubated each of them with MMR2+ EL-4 cells. Flow cytometric analysis showed HLA-A32, HLA-B13 and HLA-B62 molecules to be the ligands of human MMR2 (Table 1). Table 1 Binding of human leucocyte antigen (HLA)-A32, HLA-B13 or HLA-B62 to EL-4 cells expressing human monocyte/macrophage major histocompatibility complex (MHC) receptor (MMR)2 protein Cells . Beads . MFI (mean ± s.d.)* . MFI fold† . P‡ . MMR2+ EL-4 cells HLA (–) 50 1 ± 25·7 (n = 4) HLA-A32 690 3 ± 41·6 (n = 3) 81·2 0·00029 HLA-B13 1162 3 ± 360·5 (n = 3) 131·9 0·03755 HLA-B62 703 5 ± 10·0 (n = 4) 78·2 0·00011 HLA-B60 76 0 ± 23·8 (n = 3) 2·9 MMR2– EL-4 cells HLA (–) 41 8 ± 16·1 (n = 4) HLA-A32 16 3 ± 0·6 (n = 3) HLA-B13 67 9 ± 24·4 (n = 3) HLA-B62 54 2 ± 26·3 (n = 4) HLA-B60 51 7 ± 0·0 (n = 3) Cells . Beads . MFI (mean ± s.d.)* . MFI fold† . P‡ . MMR2+ EL-4 cells HLA (–) 50 1 ± 25·7 (n = 4) HLA-A32 690 3 ± 41·6 (n = 3) 81·2 0·00029 HLA-B13 1162 3 ± 360·5 (n = 3) 131·9 0·03755 HLA-B62 703 5 ± 10·0 (n = 4) 78·2 0·00011 HLA-B60 76 0 ± 23·8 (n = 3) 2·9 MMR2– EL-4 cells HLA (–) 41 8 ± 16·1 (n = 4) HLA-A32 16 3 ± 0·6 (n = 3) HLA-B13 67 9 ± 24·4 (n = 3) HLA-B62 54 2 ± 26·3 (n = 4) HLA-B60 51 7 ± 0·0 (n = 3) * Phycoerythrin (PE)-labelled microbeads coated with HLA-A32, HLA-B13, HLA-B62 or HLA-B60 (as a negative control) molecules were incubated with MMR2– or MMR2+ EL-4 cells, and their mean fluorescence intensity (MFI) was then analysed by fluorescence-activated cell sorter (FACS); s.d. = standard deviation. † The MFI fold = (MFI of HLA+-beads bound to MMR2+ EL-4 cells – MFI of HLA+-beads bound to MMR2– EL-4 cells)/(MFI of HLA–-beads bound to MMR2+ EL-4 cells – MFI of HLA–-beads bound to MMR2– EL-4 cells). ‡ The difference in binding between sample and control (HLA-B60) is significant according to Student's t-test. Open in new tab Table 1 Binding of human leucocyte antigen (HLA)-A32, HLA-B13 or HLA-B62 to EL-4 cells expressing human monocyte/macrophage major histocompatibility complex (MHC) receptor (MMR)2 protein Cells . Beads . MFI (mean ± s.d.)* . MFI fold† . P‡ . MMR2+ EL-4 cells HLA (–) 50 1 ± 25·7 (n = 4) HLA-A32 690 3 ± 41·6 (n = 3) 81·2 0·00029 HLA-B13 1162 3 ± 360·5 (n = 3) 131·9 0·03755 HLA-B62 703 5 ± 10·0 (n = 4) 78·2 0·00011 HLA-B60 76 0 ± 23·8 (n = 3) 2·9 MMR2– EL-4 cells HLA (–) 41 8 ± 16·1 (n = 4) HLA-A32 16 3 ± 0·6 (n = 3) HLA-B13 67 9 ± 24·4 (n = 3) HLA-B62 54 2 ± 26·3 (n = 4) HLA-B60 51 7 ± 0·0 (n = 3) Cells . Beads . MFI (mean ± s.d.)* . MFI fold† . P‡ . MMR2+ EL-4 cells HLA (–) 50 1 ± 25·7 (n = 4) HLA-A32 690 3 ± 41·6 (n = 3) 81·2 0·00029 HLA-B13 1162 3 ± 360·5 (n = 3) 131·9 0·03755 HLA-B62 703 5 ± 10·0 (n = 4) 78·2 0·00011 HLA-B60 76 0 ± 23·8 (n = 3) 2·9 MMR2– EL-4 cells HLA (–) 41 8 ± 16·1 (n = 4) HLA-A32 16 3 ± 0·6 (n = 3) HLA-B13 67 9 ± 24·4 (n = 3) HLA-B62 54 2 ± 26·3 (n = 4) HLA-B60 51 7 ± 0·0 (n = 3) * Phycoerythrin (PE)-labelled microbeads coated with HLA-A32, HLA-B13, HLA-B62 or HLA-B60 (as a negative control) molecules were incubated with MMR2– or MMR2+ EL-4 cells, and their mean fluorescence intensity (MFI) was then analysed by fluorescence-activated cell sorter (FACS); s.d. = standard deviation. † The MFI fold = (MFI of HLA+-beads bound to MMR2+ EL-4 cells – MFI of HLA+-beads bound to MMR2– EL-4 cells)/(MFI of HLA–-beads bound to MMR2+ EL-4 cells – MFI of HLA–-beads bound to MMR2– EL-4 cells). ‡ The difference in binding between sample and control (HLA-B60) is significant according to Student's t-test. Open in new tab Human MMR2 protein expression in PBMCs PBMCs or monocytes from one volunteer tested (i.e. HLA-A11,24HLA-B51,52) express both MMR1 and MMR2 mRNAs, whereas those from another volunteer (i.e. A24,33B52,61) express none [41,42]. In fact, PE-labelled HLA-B62 pentamers bound to PBMCs from the MMR2 mRNA+, but not those from the MMR2 mRNA–, volunteer within 1 min of incubation (Fig. 2); the Kd value was 8·7 × 10−9 M (Fig. 2, inset), similar to that (2·7 × 10−9 M) of the H-2Kd molecules for mouse MMR2 [40]. Fig. 2 Open in new tabDownload slide Binding of human leucocyte antigen (HLA)-B62 molecules to peripheral blood monuclear cells (PBMCs) from two volunteers, one being monocyte/macrophage major histocompatibility complex (MHC) receptor (MMR)1+ MMR2+ (open circles) and the other MMR1– MMR2– (closed circles). PBMCs were incubated with 4 × 10−8 M phycoerythrin (PE)-labelled HLA-B62 pentamers for 0–15 min. Mean fluorescence intensity (MFI) was determined by fluorescence-activated cell sorter (FACS). Each value represents the mean of two different experiments (n = 2). (Inset) Binding of HLA-B62 molecules (0–8 × 10−8 M) to PBMCs from the MMR1+ MMR2+ volunteer HLA-A11,24HLA-B51,52. MFI was determined by FACS. Each value represents the mean ± standard deviation (s.d.) of three different experiments (n = 6). Fig. 2 Open in new tabDownload slide Binding of human leucocyte antigen (HLA)-B62 molecules to peripheral blood monuclear cells (PBMCs) from two volunteers, one being monocyte/macrophage major histocompatibility complex (MHC) receptor (MMR)1+ MMR2+ (open circles) and the other MMR1– MMR2– (closed circles). PBMCs were incubated with 4 × 10−8 M phycoerythrin (PE)-labelled HLA-B62 pentamers for 0–15 min. Mean fluorescence intensity (MFI) was determined by fluorescence-activated cell sorter (FACS). Each value represents the mean of two different experiments (n = 2). (Inset) Binding of HLA-B62 molecules (0–8 × 10−8 M) to PBMCs from the MMR1+ MMR2+ volunteer HLA-A11,24HLA-B51,52. MFI was determined by FACS. Each value represents the mean ± standard deviation (s.d.) of three different experiments (n = 6). Down-regulation of mouse MMR mRNA and protein expression by expression of its ligands Mouse MMR1 (or MMR2) mRNA is expressed in PBMCs from non-H-2Dd (or non-H-2Kd; i.e. non-ligand), but not in those from H-2Dd (or H-2Kd; i.e. ligand), inbred mice [41,42]. To confirm the suppressed expression of MMR1 and 2 by their ligands expression, we transfected C57BL/6 (H-2DbKb) mice with H-2Kd and/or H-2Dd genes. As expected, transfection with the H-2Kd (or H-2Dd) transgene into these mice resulted in down-regulated MMR2 (or MMR1) mRNA and protein expression. Unexpectedly, however, it also caused down-regulation of MMR1 (or MMR2) mRNA (Fig. 3a) or protein (Fig. 3b) expression, suggesting some linkage between MMR1 and MMR2 genes. Of particular interest, therefore, H-2Dd (or H-2Kd)-transgenic mice lacking both MMR2 (or MMR1) mRNA and protein failed to reject H-2Kd (or H-2Dd)-transgenic skin (Fig. 3c). Fig. 3 Open in new tabDownload slide Down-regulated mRNA and protein expression of mouse monocyte/macrophage major histocompatibility complex (MHC) receptor (MMR)'s by expression of their ligands. (a) Agarose gel electrophoresis of reverse transcription-polymerase chain reaction (RT-PCR) products from peripheral blood monuclear cells (PBMCs) cDNAs of H-2Dd-, Kd- and DdKd-transgenic mice. M = molecular markers; WT = wild-type; W = water. Arrowheads indicate the expected size of PCR product from mouse MMR1 or MMR2 cDNA. (b) Binding of H-2 class I molecules to PBMCs from H-2Dd-, Kd- or DdKd-transgenic mice. ■ = H-2Dd molecules to PBMCs from H-2Dd-transgenic mice; □ = H-2Kd molecules to PBMCs from H-2Dd-transgenic mice; ▲ = H-2Dd molecules to PBMCs from H-2Kd-transgenic mice; △ = H-2Kd molecules to PBMCs from H-2Kd-transgenic mice. Each value represents the mean ± standard deviation (s.d.) (n = 3). (c) Survival rate of skin from H-2Kd-transgenic mice grafted onto H-2Dd-transgenic mice (●, n = 6) and that of skin from H-2Dd-transgenic mice grafted onto H-2Kd-transgenic mice (▲, n = 3). All skin grafts were tolerated. Fig. 3 Open in new tabDownload slide Down-regulated mRNA and protein expression of mouse monocyte/macrophage major histocompatibility complex (MHC) receptor (MMR)'s by expression of their ligands. (a) Agarose gel electrophoresis of reverse transcription-polymerase chain reaction (RT-PCR) products from peripheral blood monuclear cells (PBMCs) cDNAs of H-2Dd-, Kd- and DdKd-transgenic mice. M = molecular markers; WT = wild-type; W = water. Arrowheads indicate the expected size of PCR product from mouse MMR1 or MMR2 cDNA. (b) Binding of H-2 class I molecules to PBMCs from H-2Dd-, Kd- or DdKd-transgenic mice. ■ = H-2Dd molecules to PBMCs from H-2Dd-transgenic mice; □ = H-2Kd molecules to PBMCs from H-2Dd-transgenic mice; ▲ = H-2Dd molecules to PBMCs from H-2Kd-transgenic mice; △ = H-2Kd molecules to PBMCs from H-2Kd-transgenic mice. Each value represents the mean ± standard deviation (s.d.) (n = 3). (c) Survival rate of skin from H-2Kd-transgenic mice grafted onto H-2Dd-transgenic mice (●, n = 6) and that of skin from H-2Dd-transgenic mice grafted onto H-2Kd-transgenic mice (▲, n = 3). All skin grafts were tolerated. Binding of ≤ 10 types of HLA-conjugated microbeads to monocytes from two representative volunteers, one expressing both MMR1 and MMR2 and the other expressing none Humans are typically outbred mammals, and a fetus expresses paternal MHC class I molecules in addition to maternal molecules. In addition, an earlier study showed that none of 16 healthy volunteers express HLA-B44, a ligand of MMR1, whereas MMR1 mRNA is expressed only in PBMCs from three of 16 volunteers [41], implying down-regulation of MMR1 expression by some linkage among human MMR genes, as described above in the mouse (Fig. 3). Next, we explored how many types of HLA-conjugated microbeads bound to CD14+ PBMCs or monocytes from two representative volunteers, one expressing both MMR1 and MMR2 and the other expressing neither (Fig. 4). As expected, microbeads conjugated with HLA-B44, B13 or B62 bound specifically to monocytes from the volunteer (i.e. HLA-A11,24HLA-B51,52) expressing both MMR1 and MMR2, because B44 is the sole ligand of MMR1 [41] and because both B13 and B62 are ligands of MMR2 (Fig. 4a). In addition, HLA-B35 and B60 bound significantly to the monocytes; the binding of HLA-B54-coated microbeads was less significant. HLA-B45 appeared to bind to the monocytes, whereas the binding was not significant (P = 0·05684). HLA-A32 was one of the ligands for MMR2 (Fig. 1 and Table 1), whereas there was no binding of HLA-A32-conjugated microbeads to the monocytes, suggesting that HLA-A32 molecules might bind only to MMR2 cDNA-transfected cells expressing a large amount of human MMR2 proteins. Microbeads conjugated with HLA-A11, A-24, B51 and B52, however, did not bind to the monocytes because they were self-MHC. These results indicate that monocytes from the MMR1+/MMR2+ volunteer expressed four to six types of MMRs for seven types of HLA class I molecules: MMR1 for HLA-B44, MMR2 for HLA-B13 and B62, and two to four types of MMRs for HLA-B35, B45, B54 and B60. Fig. 4 Open in new tabDownload slide Binding of beads conjugated with various human leucocyte antigen (HLA) class I proteins to monocytes from two, monocyte/macrophage major histocompatibility complex (MHC) receptor (MMR)1+MMR2+ (a) and MMR1–MMR2– (b), human volunteers. The mean fluorescence intensity (MFI) fold was determined as described in the legend of Fig. 1. Each value represents the mean ± standard deviation (s.d.) of three different experiments (n = 6). (a) HLA-A11,24HLA-B51,52. The difference in binding between sample and control (HLA-A24: self-MHC) is significant (*P < 0·02; **P < 0·005; ***P < 0·002; ****P < 0·001) according to Student's t-test. (b) HLA-A24,33HLA-B52,61. The difference in binding between sample and control (HLA-A33: self-MHC) is significant (*P < 0·02; **P < 0·0002) according to Student's t-test. Fig. 4 Open in new tabDownload slide Binding of beads conjugated with various human leucocyte antigen (HLA) class I proteins to monocytes from two, monocyte/macrophage major histocompatibility complex (MHC) receptor (MMR)1+MMR2+ (a) and MMR1–MMR2– (b), human volunteers. The mean fluorescence intensity (MFI) fold was determined as described in the legend of Fig. 1. Each value represents the mean ± standard deviation (s.d.) of three different experiments (n = 6). (a) HLA-A11,24HLA-B51,52. The difference in binding between sample and control (HLA-A24: self-MHC) is significant (*P < 0·02; **P < 0·005; ***P < 0·002; ****P < 0·001) according to Student's t-test. (b) HLA-A24,33HLA-B52,61. The difference in binding between sample and control (HLA-A33: self-MHC) is significant (*P < 0·02; **P < 0·0002) according to Student's t-test. In the case of the volunteer (i.e. HLA-A24,33HLA-B52, 61) expressing neither MMR1 nor MMR2, microbeads conjugated with HLA-A23, A26, A68 or B50 bound significantly to CD14+ PBMCs or monocytes (Fig. 4b). In addition, the binding of HLA-A2-, A11-, A69-, B13- or B81-coated microbeads to the MMR1–/MMR2– monocytes was less significant. As HLA-A32 and B62 did not bind to the monocytes, HLA-B13, a ligand of MMR2, might bind to MMR(s) other than MMR2. Microbeads conjugated with HLA-A24, A-33, B52 and B61, however, did not bind to the monocytes because they are self-MHC. Microbeads conjugated with HLA-A32, B44 and B62 did not bind to the monocytes because they are ligands of MMR1 and MMR2. These results indicate that monocytes from the MMR1–/MMR2– volunteer expressed three to nine types of MMRs other than MMR1 or MMR2 for nine types of HLA class I molecules. Discussion It has been recognized that CTLs, which include ≥ 1018 (=5·8 × 106 pairs of V genes × 2 × 1011 junctional diversity) sets of T cells expressing a type of T cell receptor (TCR) reactive with a non-self peptide in association with an HLA class I molecule [49], are the effector cells responsible for allograft rejection [13–16]. Conversely, we found that human MMR1 and MMR2 were specific receptors for HLA-B44 [41] and HLA-A32, B13 and B62 (Figs 1 and 2 and Table 1) molecules, respectively; that H-2Kd (or H-2Dd) transgene into C57BL/6 mice down-regulated not only MMR2 (or MMR1), but also MMR1 (or MMR2) expression (Fig. 3); and that monocytes from two (MMR1+/MMR2+ and MMR1–/MMR2–) volunteers expressed three to nine types of MMRs towards seven to nine types of HLA class I molecules among the 80 molecules (Fig. 4) covering 99·4% and 96·2% of HLA-A and B molecules in Japanese individuals [46]. Thus, receptor ligand mechanisms for allorecognition by ≤ 10 types of MMRs on innate cells (i.e. monocytes) were quite distinct from those of antigen recognition by 1018 sets of TCRs on adaptive cells (i.e. CTLs). Sequence studies indicate that 10 types of MHC class I heavy chains are among the most polymorphic molecules encoded in the mouse genome [50], whereas only one type of receptor, MMR1 or MMR2, for one to three allogeneic MHC class I molecules was expressed constitutively on mouse or human monocytes ([39–42], Fig. 1 and Table 1). The Kd (1·9–8·9 × 10−9 M) of MHC class I molecules for MMR1 or MMR2 on AIM [39–42] or monocytes (Fig. 2) was much lower than that (10−7 M; [51]) of the H-2Ld molecule towards TCR. In addition, the MMR-mediated allorecognition was MHC-unrestricted ([25,26]; Figs 1, 2 and 4 and Table 1), whereas TCRs recognize antigens in an MHC-restricted manner [52]. These results suggest that the mechanisms of monocyte/macrophage-mediated allorecognition might be basically quite distinct from those of lymphocyte-mediated antigen recognition. Therefore, it is reasonable that the binding region of MHC class I molecules to MMR1 or MMR2 [39–42] has no homology with immunoglobulins (Igs) [53], T cell receptors [54], natural killer Ig-like receptors [55] or the leucocyte Ig-like receptor family [56]. In the case of human MMR1, 12·6% of Native Americans and 7·4% of Japanese have HLA-B44 [45,46], which is the sole ligand of human MMR1 [41]. A homology search of the amino acid sequences of HLA molecules showed that the HLA-B44 had ≥ 94% amino acid identity to HLA-47, B13, B53, B49, B37, B40, B45, B27 or B57 molecules, and suggested that the binding site might include the amino acid sequence at positions 137–140 in the α2 region [41]. In the case of human MMR2, 2·55, 1·75 and 6·94% of Native Americans and 0, 1·48 and 7·06% of Japanese have HLA-A32, B13 and B62, respectively [45,46], all of which were the ligands of human MMR2 (Figs 1–3). Serologically, HLA-A32 is included in a group of HLA-A19, which also contains HLA-A19, A29, A30, A31, A33 and A74 [45], but human MMR2 reacted exclusively with HLA-A32 (Fig. 1). In addition, HLA-B62 is classified serologically as a group of HLA-B15 including HLA-B63, B70, B71, B72, B75, B76 and B77 [45], whereas HLA-B62 bound specifically to human MMR2 (Fig. 1). Of particular interest, there is 97·8, 84·5 and 84·0% amino acid identity between HLA-B62 and HLA-B71, HLA-B13 or HLA-A32 molecules, respectively, whereas MMR2 reacted with the latter two HLA class I molecules (Figs 1 and 2 and Table 1), suggesting that the mechanism of HLA class I recognition by MMR2 might depend upon the secondary or tertiary structure. Mouse MMR2 mRNA is expressed in PBMCs from non-H-2Kd (i.e. non-ligand), but not H-2Kd (i.e. ligand), inbred mice [42]. Similarly, mouse MMR1 mRNA is expressed in PBMCs from non-H-2Dd (i.e. non-ligand), but not H-2Dd (i.e. ligand), inbred mice [41]. H-2Kd (or H-2Dd) transgene into C57BL/6 (H-2DbKb) mice down-regulated not only MMR2 (or MMR1) but also MMR1 (or MMR2) expression (Fig. 3). In fact, PBMCs from four of 16 volunteers expressed HLA-B62 molecules (a ligand for MMR2), whereas PBMCs from these 16 volunteers expressed MMR2 with a frequency of eight of 12 volunteers [42]; none of the PBMCs from the 16 volunteers expressed HLA-B44 molecules (a ligand for MMR1), whereas PBMCs from these 16 volunteers expressed MMR1 with a frequency of three of 16 [41]. Furthermore, MMR2 knock-out mice failed to reject skin grafts from H-2Dd- or H-2DdKd-transgenic mice as well as from H-2Kd-transgenic mice, but they rejected skin grafts from mice expressing H-2Id, minor Hd or third-party MHC [44]. Therefore, these results suggest that there might be some linkage between MMR1 and MMR2 genes and that down-regulated expression of MMR1 and MMR2 was ligand-specific. MMRs including MMR1 and MMR2 are not expressed on resident macrophages in the peritoneal cavity, and when allogeneic Meth A (H-2d) cells are transplanted i.p. into C57BL/6 mice, the expression of both MMR1 and MMR2 is induced in allograft (H-2d)-induced macrophages (H-2b) on days 5–12 after transplantation [25,26]. Another study showed that the macrophages (H-2b) exhibit H-2d haplotype-specific cytotoxic activity against allografts: AIM attach tightly to a Meth A cell with a Kd of 1·9∼2·7 × 10−9 M [39,40], seem to ‘bite off’ a fragment of the plasma membrane expressing MHC molecules, and then detach themselves from the target cell [37]. As monocytes have much higher capacity to release reactive oxygen intermediates including H2O2 than do macrophages [57], we expected reactive oxygen intermediate-mediated cytotoxic activity of MMR2+ monocytes against HLA-B62+ cells. However, preliminary experiments from our laboratory demonstrated that relative fluorescence from dichlorodihydrofluorescein diacetate (H2DCFDA)-labelled PBMCs expressing MMR2 was suppressed dose-dependently by the addition of HLA-B62 pentamer, a ligand of human MMR2 (Fig. 2), with half maximal inhibitory concentration (IC50) of 5·5 × 10−9 M, implying a mechanism of down-regulation of MMR expression by the expression of self-MHC. Moreover, H-2Kd (or H-2Dd)-transgenic C57BL/6 mice showed down-regulation of not only MMR2 (or MMR1) but also MMR1 (or MMR2) expression (Fig. 3), causing further decrease in the types of MMRs expressed on monocytes. After all, monocytes from the two volunteers, one expressing both MMR1 and MMR2 and the other expressing neither, bound only seven to nine types of HLA class I molecules among the 80 types examined (Fig. 4), suggesting a tolerance of MMRs towards a large number of HLA class I molecules. Similarly, during the reproductive period, mother and offspring exchange haematopoietic cells and develop a long-lasting form of the maternal (or fetal) tolerance to fetal (or maternal) HLA antigens of inherited paternal (or non-inherited maternal) origin [58]. Therefore, the physiological role of constitutive MMRs on monocytes possibly towards allogeneic (e.g. fetal) cells in the blood appears to be quite distinct from that of inducible MMRs on macrophages towards allografts in tissue. Acknowledgements We thank T. Ueno, R. Oide and Y. Nakahira for their skillful technical assistance. This work was supported in part by the programme Grants-in-Aid for Scientific Research (C) (grant no. 20591538) and Young Scientists (B) (grant no. 23791505) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Mori and Magari Memorial Research Funds from Osaka Medical College. Disclosures The authors have no financial conflicts of interest. Author contributions H. Y., J. T.-Y., S. M., T. S. and R. Y. participated in the performance of the research. H. Y., J. T.-Y., M. H. and R. Y. participated in the data analysis of the research. H. Y., J. T.-Y., M. H., N. T., K. U., T. K. and R. Y. participated in the research design and writing of the article. References 1 Beutler B , Milsark IW, Cerami AC. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin . Science 1985 ; 229 : 869 – 871 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Akira S , Nishio Y, Inoue M et al. 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Google Scholar Crossref Search ADS PubMed WorldCat © 2014 British Society for Immunology This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Down-regulated expression of monocyte/macrophage major histocompatibility complex receptors in human and mouse monocytes by expression of their ligands
JF - Clinical & Experimental Immunology
DO - 10.1111/cei.12383
DA - 2014-09-04
UR - https://www.deepdyve.com/lp/oxford-university-press/down-regulated-expression-of-monocyte-macrophage-major-JDjkO0Ij0s
SP - 118
EP - 128
VL - 178
IS - 1
DP - DeepDyve
ER -