, forward scatter
side scatter MICL: myeloid inhibitory C-type lectin
C-type lectins are the most diverse and prevalent lectin family in immunity. Particular interest has recently been attracted by the C-type lectin-like receptors on NK cells, which appear to regulate the activation/inhibitory balance of these cells, controlling cytotoxicity and cytokine production. We previously identified a human C-type lectin-like receptor, closely related to both the beta-glucan receptor and the lectin-like receptor for oxidized-LDL, named MICL (myeloid inhibitory C-type lectin-like receptor), which we had shown using chimeric analysis to function as an inhibitory receptor. Using a novel MICL-specific monoclonal antibody, we show here that human MICL is expressed primarily on myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Although MICL was highly N-glycosylated in primary cells, the level of glycosylation was found to vary between cell types. MICL surface expression was down-regulated during inflammatory/activation conditions in vitro, as well as during an in vivo model of acute inflammation, which we characterize here. This suggests that human MICL may be involved in the control of myeloid cell activation during inflammation.
In order to execute their immune (and non-immune) functions, leukocytes must interact with a broad range of endogenous and exogenous ligands and must be able to respond to these interactions appropriately. This requires a wide and flexible, yet specific and regulated, repertoire of cell surface molecules. One large family of such molecules, which are particularly important to immunity, are the C-type lectin and lectin-like receptors. C-type lectin-like receptors were first identified as dimeric molecules on NK cells. On these cells, much attention has been drawn toward their ability to regulate the balance between cellular activation and inhibition, such as cytotoxicity and cytokine production. This is achieved through signalling via intracellular ITIM present in the cytoplasmic tails of these receptors or via ITAM, of which the majority are located in the cytoplasmic tails of associated molecules, such as DAP12. The study of these activation and inhibitory NK cell receptors has generated a number of interesting hypotheses, including immune privilege 1, the recognition of ‘missing self’, ‘non-self’ and ‘induced self’ 2–5, as well elucidating the underlying mechanisms of some of the immune responses to viruses 6 and malignancy 7.
Others and we have demonstrated that C-type lectin-like receptors are not restricted to NK cells, but are expressed by many other cell types including myeloid cells 8–10. This introduces the novel concept that the myeloid paralogs of NK cell receptors might govern myeloid cell activation/inhibition in the same manner as those on NK cells. The genes of most C-type lectin-like molecules are located within the ‘natural killer complex’ (NKC), but many of those expressed by myeloid and other cells are found in a distinct cluster of genes within the NKC 10. This cluster includes Dectin-1/ beta-glucan receptor 11, the lectin-like receptor for oxidized LDL (LOX-1) 12, CLEC-1 and CLEC-2 13, two unstudied transcripts 14, and myeloid inhibitory C-type lectin-like receptor (MICL) 14.
MICL (also called CLL-1, KLRL1 and DCAL-2) has been independently identified by several groups and shown to be a type II transmembrane receptor comprising a single C-type lectin-like domain (which is not predicted to bind either calcium or sugar), a stalk region, a transmembrane domain and a short cytoplasmic tail containing an ITIM motif 14–17. MICL orthologs have been identified in the mouse, rat, and dog, indicating that the receptor is conserved across species. MICL can be alternatively spliced into at least three isoforms and is highly N-glycosylated when expressed in heterologous cell lines. Others and we have shown that MICL can associate with the inhibitory signalling phosphatases, SHP-1 and SHP-2, and that MICL can function as an inhibitory receptor 14–16. The ligand of this receptor is unknown.
In this study, we have generated a novel anti-MICL mAb and used it to characterize the expression and function of MICL in primary human cells. We demonstrate that MICL is expressed predominantly by neutrophils, eosinophils, monocytes, MΦ, and DC. This receptor was also present at low levels on CD4+ T cells but was not expressed by NK cells or other lymphocytes. We show that MICL is differentially glycosylated and is down-regulated during inflammation/cellular activation, both in vitro and in vivo, using a model of acute inflammation, which is characterized here.
A new mAb, HB3, specifically recognizes human MICL
To investigate MICL protein expression and function in primary human cells, we generated a mouse mAb specific to this receptor. An Fc-MICL fusion protein was used to immunize C57BL/6 mice and hybridomas were generated and screened, as described in the Materials and methods. One particular mAb, HB3 (IgG1), was chosen for further analysis as it could be utilized in a variety of applications, including flow cytometry, Western blotting, and immunochemistry (Fig. 1).
To confirm that HB3 is specific for MICL, its reactivity against various MICL-containing reagents was tested. By flow cytometry, HB3 reacted with NIH3T3 cells transduced to express HA-tagged MICL but not with control cells, and this reactivity was not significantly affected by fixation (Fig. 1A and data not shown). In Western blot analysis, under non-reducing conditions, HB3 specifically recognized a range of protein sizes, resolving at 40–75 kDa, from lysates of HA-tagged MICL-transduced cells (Fig. 1B). These sizes were comparable to those obtained when the membranes were probed with an anti-HA mAb (data not shown and see Marshall et al. 14). Protein bands corresponding to MICL were also detected with HB3 in lysates from CHO-lec-1 transductants (N-glycosylation deficient) and a bacterially expressed MICL construct, indicating that glycosylation of the MICL polypeptide is not required for, nor affects recognition by this mAb (data not shown). However, some degree of folding may be required, as denatured MICL, under reducing conditions, could not be detected by HB3. Finally, HB3 was confirmed to be specific for MICL by immunocytochemistry with paraformaldehyde-fixed transductants (Fig. 1C).
The distribution of MICL within peripheral blood and selected tissues
Using HB3, we next characterized MICL protein expression on PBL by flow cytometry. Following erythrocyte lysis, blood leukocytes were stained with HB3 in combination with various markers of monocytes, granulocytes and lymphocytes. These three major leukocyte populations were distinguishable by size and granularity (Fig. 2A). Granulocytes, which were gated by high side scatter (SSChi) and confirmed to be CD15+ (data not shown), clearly expressed MICL (Fig. 2A; R1). Neutrophils, which form the majority of blood granulocytes, were evidently MICL-positive, and eosinophils, gated by their high autofluorescence 18, expressed MICL to similar levels (data not shown). Monocytes, gated by high forward scatter (FSChi)SSClo, were also MICL-positive (Fig. 2A; R2). Blood monocytes can be divided into those that are recruited to inflammatory sites (CD14+16–; R5) and those that are constitutively recruited to become tissue resident MΦ and DC (CD14low16+; R4) 19. Both these populations expressed MICL to equivalent levels, as did double-negative cells (which may represent peripheral blood DC) and double-positive cells (Fig. 2B; R6 and data not shown). More detailed examination of DC, demonstrated that both the myeloid (CD1c+CD19–) and BDCA2+ plasmacytoid DC expressed MICL on their cell surface (Fig. 2C). Lymphocytes, which were gated by FSCloSSClo, appeared largely MICL negative although there was a slight positive shift on the gated population (Fig. 2A; R3). Using specific markers to subdivide the lymphocytes, NK cells, NKT cells, B cells and CD8+ T cells were all shown to be MICL negative, however, CD3+CD4+ T cells consistently expressed low levels of MICL (Fig. 2D). Thus, MICL expression is predominantly restricted to myeloid cells within peripheral blood.
To confirm our findings in peripheral blood, HB3 was used to screen spleen and lymph node tissue for MICL expression (Fig. 3). Spleen sections displayed strong MICL expression in the MΦ-rich red pulp. Some MICL-positive cells were also observed in the white pulp, which may represent MΦ or DC. Lymph nodes displayed a similar MICL distribution, with high levels in the MΦ-dense medullary sinuses and less in the follicles. Thus, these data support the hypothesis that MICL expression is largely confined to myeloid cells.
MICL expression and regulation on in vitro cultured MΦ and DC
As expression of C-type lectins can be affected by the stage of cellular differentiation 20, we next examined MICL expression during in vitro differentiation of peripheral blood monocytes into MΦ and DC, as described in the Materials and methods. Cells were analyzed at various time points by flow cytometry using anti-MICL (HB3) and other myeloid markers (CD14, DC-SIGN and CD86) to confirm their phenotype (Fig. 4A). On cultured MΦ, MICL expression remained stable during differentiation and did not decrease, as was predicted from previous mRNA analysis 14. In fact, MICL expression was stable on cultured MΦ analysed up to day 16 (data not shown). Compared with surface levels on these cells, intracellular pools of this receptor were negligible (data not shown). In DC, MICL expression remained similarly equivalent during differentiation from monocytes, induced by IL-4 and GM-CSF. However, activation of DC with LPS induced a marked down-regulation of this receptor (Fig. 4A) which could be reproduced, albeit to a lesser extent, on cultured MΦ following LPS treatment (data not shown).
To address this further, we stimulated freshly isolated monocytes with TLR agonists and examined their effect on MICL expression. As shown in Fig. 4B, stimulation with TLR2/TLR1 (pam3csk4), TLR2/TLR6 (pam2csk4) and TLR4 (LPS) ligands, significantly reduced the level of MICL expression on the cell surface. By examining the effect of LPS treatment on MICL mRNA levels by RT-PCR (Fig. 4C), we observed that the down-regulation of MICL appeared to be occurring at the level of transcription.
MICL expression during inflammation in vivo
As MICL appeared to be generally down-regulated following cellular activation in vitro, as described above, we sought to determine whether MICL might be similarly regulated in an in vivo inflammatory setting. For this analysis, we utilized an acute inflammatory model, as detailed in the Materials and methods, to allow a comparison of MICL expression on cells recruited to the site of inflammation, with those in peripheral blood. Cells harvested from the inflammatory focus were examined by flow cytometry and found to consist predominantly of two major populations, which corresponded to peripheral blood granulocytes and monocytes by size and granularity (Fig. 5A). As there is little data available on the characteristics of ex vivo inflammatory human cell populations, we characterized this model further, using a range of antibodies, to identify markers of the different cellular populations (Fig. 5B). On the recruited granulocyte population (R1; 59% of total), CD15 expression remained unchanged, in comparison with peripheral blood (data not shown), whereas CD16 expression was down-regulated on most recruited cells (Fig. 5B). The recruited monocytes/MΦ (R2; 8% of total) displayed up-regulation of both CD86 and HLA-DR (neither of which were expressed on the recruited or peripheral blood granulocytes), down-regulation of CD14 and an absence of CD16 expression (Fig. 5B and data not shown). CD11b was up-regulated on both the recruited granulocytes and monocytes/MΦ. By CD15/CD86 staining, MΦ (R2; CD86+15–; 7% of total) could easily be distinguished from recruited granulocytes (R1; CD86–15+; 70% of total), and were confirmed to be distinct but overlapping populations by size and granularity (Fig. 5C). Recruited cells of very high FSC and SSC, shown in Fig. 5A, displayed high autofluorescence and were not included in this analyses. However, when analyzed separately, their expression of the various markers, including MICL, was not noticeably different from that of the recruited granulocyte population (data not shown).
Using CD15/CD86 staining to distinguish the two major populations, we examined MICL expression on these cells after 48 h and found that the receptor was consistently down-regulated on both recruited granulocytes and monocytes/MΦ (Fig. 6A and B). Further analysis of MICL expression on the recruited granulocytes, demonstrated that down-regulation of this receptor was concomitant with the reduction in CD16 expression, as described above. This down-regulation of MICL was specific, as other receptors on these cells, including Dectin-1 and EMR1, were regulated differently (21 and data not shown). At 24 h, when some recruited granulocytes still maintained CD16 expression, two subpopulations (CD16+MICL+ and CD16–MICL–) were easily definable, although some background nonspecific staining was observed with the CD16 isotype control (Fig. 6C). Overall, our data demonstrate that MICL is down-regulated on activated leukocytes following recruitment to a focus of acute inflammation in vivo.
Glycosylation of MICL in primary cells
We have previously noted that MICL was highly glycosylated when expressed in heterologous cell lines, and we wished to confirm these observations in primary cells. Using the anti-HB3 mAb to detect MICL in cellular lysates, we observed that a range of proteins were detected with molecular mass between 40 and 75 kDa in neutrophils and mononuclear cells, but not in erythrocytes, which corresponded in size to those seen in transfected cells (Fig. 7). Intriguingly, in cultured monocytes, the molecular mass of the MICL-positive bands appeared to increase over time, depending on the stage of differentiation, although the overall levels of receptor on the cell surface did not appear to change (Fig. 7).
To determine whether these discrepancies in MICL molecular mass were due to the expression of different splice variants 14 or to variable glycosylation, cell lysates were de-glycosylated and examined by Western blotting (Fig. 7). PNGase F, which completely removes N-linked glycans, was used to treat cell lysates from non-adherent mononuclear cells and all cultured monocyte/Mϕ populations. A predominant de-N-glycosylated MICL-positive band was observed at ∼30 kDa, indicating that variable glycosylation, not alternative splicing, was responsible for the differing molecular mass observed in the untreated samples. Thus, the level of MICL glycosylation is regulated in different cell types. From this analysis, it also appears that MICL may be functioning as a monomer, as higher molecular mass complexes were not observed.
C-type lectin-like receptors, such as those expressed on NK cells, play important roles in regulating immune and non-immune functions of lymphocytes. These receptors appear to recognize protein ligands, mostly MHC class I or related molecules, triggering activation or inhibitory intracellular signals. Similar receptors have also been identified on myeloid cells and although those that have been characterized appear to recognize a more diverse range of molecules 20, the ligands of many are unknown. One such “orphan” is MICL, a group V C-type lectin-like receptor that we originally identified based on homology to Dectin-1 14. MICL contains an ITIM in its cytoplasmic tail and our initial studies using chimeras suggested that this molecule could function as an inhibitory receptor. To gain more insight into the role of this receptor, we generated a novel mAb and have used it here to examine MICL on primary cells.
Our analysis of MICL, in both transduced cell lines 14 and primary cells, suggests that this molecule is highly N-glycosylated compared to its most closely related paralogs, lectin-like receptor for oxidized LDL 22, Dectin-19, 23, and CLEC-2 13. Intriguingly, the level of MICL N-glycosylation varied significantly in different leukocyte populations, which may have functional consequences for ligand binding, as shown for other receptors 24, 25. Many other group V receptors dimerize through cysteines in the stalk region (see http://ctld.glycob.ox.ac.uk/), and although MICL contains the necessary residues 14, we have not been able to detect multimerised forms of this receptor in primary cells. However, we 14 and others 16 have observed higher molecular weight forms in transfected cells and cell lines, which may indicate that homo-/hetero-multimerisation of MICL can occur. While predominant monomeric forms for other NK lectin-like receptors, such as KLRI126, KLRI226 and KLRE127, have been observed, it is possible that intracellular signalling requires homo-/hetero-dimerisation 27.
MICL expression was predominantly restricted to myeloid cells in both peripheral blood and in tissues, and although low levels were observed on CD4+ T cells, this receptor was not detected on any other lymphocyte population. This expression profile of MICL is similar to that of the related receptor, beta-glucan receptor/Dectin-1, found in the same gene cluster 21, and is largely consistent with previous transcript analyses 14, 16, 17, although some differences were noted, as discussed above. MICL has also been proposed to be expressed on NK cells, based on the ability of an Fc-MICL fusion protein to inhibit cytotoxicity 16. However, our data, and those of others 17, clearly demonstrate that MICL is absent from these cells. Indeed, we have found that Fc-C-type lectin-like fusions often bind nonspecifically to lymphocytes (data not shown), perhaps explaining these observations. Consistent with a myeloid restricted profile, MICL has been detected in more than 90% of acute myeloid leukaemia (AML) blast cell samples 17.
To gain more insight into the function of MICL on primary myeloid cells, we utilized an in vivo model allowing the isolation and analysis of inflammatory cells. A number of other models of aseptic inflammation in humans have been developed, including: ‘skin windows’, involving removal of the stratum corneum by a blade 28, drill 29, or by tape stripping 30; suction blisters 31; and blisters induced by cantharidin 32. In addition, some of these techniques have involved the collection of recruited cells in a chamber containing a chemoattractant 29, 31, 33. The method we used here, involving abrasion of the skin with sand paper, is similar to the other ‘skin window’ models, but with the benefit that it is simple and easy to perform and contains fewer contaminating erythrocytes 29. As in other models 32, the major recruited populations were granulocytes followed by monocyte/MΦ-like cells. Some techniques appear to attract a small number of lymphocytes 32, but these were not observed in our model.
Although few models have characterized the surface markers on recruited cells, our analysis is comparable with other in vitro and in vivo methods of cellular activation. The down-regulation of FcγRIII (CD16) has been observed on granulocytes that have been stimulated with fMLP 34, transmigrated across cultured epithelium 35 and on granulocytes undergoing apoptosis 36. Recruited monocytes/MΦ also did not express CD16 in our model, corroborating the hypothesis that peripheral blood CD16+ monocytes are not recruited to inflammatory sites 19, 37, 38. The up-regulation of adhesion molecules, such as CD11b, has similarly been observed following both granulocyte and monocyte activation 39, 40, and CD86 and MHC class II are recognized markers of monocyte/MΦ activation 41. The down-regulation of molecules such as CD14, on monocytes/MΦ has also been previously reported 42–44. Some minor differences with other models were, however, noted. For example, in cantharidin blisters, some recruited cells were CD16+ or CD14+32, but these cells were not detected in our model. Overall, this method provides an easy in vivo model for the analysis of human inflammatory cells.
Using this model, as well as in vitro analysis, we observed that expression of MICL was down-regulated upon cellular activation, consistent with the hypothesis that activation can be facilitated by removal of inhibitory molecules. Although the exact functions of this receptor are unknown, as are roles of many inhibitory receptors, MICL may play an role in the control of cellular maturation 15 and activation. Indeed loss of inhibitory receptor function has been implicated in a number of diseases that involve cellular activation, including autoimmunity 45 and allergy 46. Using antibody cross linking, we have also examined the possibility that MICL may be involved in apoptosis, as has been demonstrated for other inhibitory receptors 47–49, but despite considerable effort we were unable to demonstrate a role for MICL in this process (data not shown). It seems likely therefore that the functions of this receptor will only be clearly established following the identification of its ligand.
In summary, we have shown that the expression of the inhibitory receptor MICL is predominantly restricted to myeloid cells. This receptor is differentially glycosylated in various cell types and is down-regulated following cellular activation. This suggests that MICL may be involved in the control of myeloid cell activation.
Materials and methods
Generation of mAb against MICL
The mAb, HB3, specific for MICL, was generated by immunization of C57BL/6 mice with an Fc-MICL fusion protein. The Fc-MICL expression construct was generated by PCR and cloned into the pSecTag2 vector (Invitrogen) upstream of an ‘Fcmut’ construct, described previously 50. Hybridomas were generated according to standard protocols 51, 52, and supernatants from clonally diluted cells were screened by ELISA. The mAb HB3 (IgG1) was subsequently selected based on its ability to function in FACS, Western blot, and immunocytochemistry. HB3 was biotinylated using a protocol provided by S. Cobbold (see http://users.path.ox.ac.uk/∼scobbold/tig/fitc.html).
Cells and cell culture
The generation of NIH3T3 cells expressing HA-tagged MICL has been described previously 14. These cells were maintained in DMEM supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM L-glutamine (DMEM medium) and 0.6 mg/mL G418.
To isolate PBL for flow cytometric analysis, peripheral blood from healthy volunteers was collected into 4mM EDTA, and the erythrocytes lysed with Gey's solution. The cells were then layered onto 100% FCS and the intact leukocytes were collected by centrifugation and analyzed, as described below.
For in vitro monocyte culture, PBMC were isolated from buffy coats using a gradient of Ficoll-HypaqueTM PLUS (Amersham), according to standard protocols. Adherent monocytes, isolated as described previously 21, were cultured in X-VIVO medium (BioWhittaker) with 1% heat-inactivated autologous serum for differentiation into MΦ, or cultured in RPMI with 10% heat-inactivated FCS, 50 ng/mL GM-CSF and 25 ng/mL IL-4 (R&D Systems) to produce DC. Activation/maturation of immature DC (day 6) was achieved with the addition of 1 μg/mL LPS for 48 h.
For analysis of the effects of TLR agonists on MICL expression, freshly isolated adherent monocytes were incubated with LPS (1 μg/mL; Sigma), Pam2CSK4 (100 ng/mL; InvivoGen) or Pam3CSK4 (100ng/mL; InvivoGen) for 36 h. The effects on MICL expression were analysed using immunofluorescence flow cytometry, as described below, or by RT-PCR. In brief, RNA was extracted from LPS-treated or untreated monocytes, using the Total RNA Isolation System (Promega, WI). cDNA was synthesized using Advantage RT-for-PCR Kit (BD Biosciences, CA), according to the manufacturer's specifications, and PCR was performed using MICL-specific primers (5′-AAA CCC GGG TCT GAA GAA GTT ACT TAT GC-3′ and 5′-AAA CCC GGG AAT GTA TTT GAT TGA TGC C-3′) or human G3PDH control primers (BD Biosciences, USA).
To isolate peripheral blood populations for Western blotting, heparinised blood from healthy volunteers was layered over a solution of PolymorphPrep (Nycomed Pharma AS, Oslo, Norway) and centrifuged at room temperature (RT) at 500 × g for 35 min. The total mononuclear (top; including monocytes and lymphocytes), polymorphonuclear (middle) and erythrocyte (bottom) fractions were collected separately, washed extensively and analyzed by Western blotting as described below.
All experiments were repeated on cells isolated from at least three different donors.
In vivo inflammatory model
To provide an inflammatory model for study of activated human leukocytes, small abrasions (2 cm2) were generated on the forearm skin of volunteers with sand paper until a small amount of capillary bleeding was observed. Informed consent was provided according to the Declaration of Helsinki. A filter paper strip, pre-soaked with sterile PBS, was placed on the abraded area and sealed overnight. The strip was replaced with fresh filter paper and left for another 24 h before removal and analysis. Recruited cells were released from the filter paper with PBS/EDTA, harvested by low speed centrifugation at 200 × g, to minimize contamination with dead cells and cell debris, and then analyzed by flow cytometry, as described below. The difference in MICL expression between peripheral blood and recruited populations, using median fluorescence values, was calculated as follows: (MICL staining on recruited cells/isotype control on recruited cells)/(MICL staining on peripheral cells/isotype control on peripheral cells). All experiments were repeated on at least three different donors.
Antibodies and flow cytometry
Cells were analyzed by three-color flow cytometry according to standard protocols in the presence of 2 mM NaN3 at 4°C. Cells were blocked in PBS containing 5 mM EDTA, 0.5% BSA, and either 5% heat-inactivated rabbit serum (NIH3T3) or murine serum (PBL) prior to the addition of the primary antibodies. Antibodies used in these experiments included: HB3 and HB3-biotin, GE2 and GE2-biotin 21, CD4 (CFAR), CD14-FITC (BD PharMingen), CD16 (Serotec), CD16-PE (BD PharMingen), CD56-PE (BD PharMingen), CD19-PE (BD PharMingen), DC-SIGN-FITC (BD PharMingen), CD19-FITC (Caltag), CD1c (Serotec), BDCA253, CD8-PE (Serotec), CD83-PE (Serotec), CD86-PE (Serotec), CD3-FITC (Serotec), CD11b-FITC (Serotec), HLA-DR-FITC (Serotec), CD15-FITC (Serotec), and irrelevant unlabelled or biotin-, PE- or FITC-labelled mouse or rat IgG1 (including D1.3; a gift from L. Martinez-Pomares, Oxford University, Oxford, UK), IgM (Serotec) and IgG2b (ECACC) control antibodies. Biotinylated antibodies were detected using streptavidin-allophycocyanin (BD PharMingen). Cells were fixed with 1% formaldehyde, 0.25% BSA in PBS before analysis.
Western blotting and de-glycosylation
To prepare cellular extracts, washed cells were lysed in an NP-40 buffer (1% NP-40, 0.15 M NaCl, 10 mM EDTA, 10 mM NaN3, 10 mM Tris-HCl pH 8), supplemented with protease inhibitors (Roche), for 30 min at 4°C. Cellular debris/nuclei were removed by centrifugation and supernatants were collected and stored at –20°C. Protein concentrations of cell extracts were determined by the BCA assay (Pierce) and equal quantities of protein were run on SDS-PAGE gels, according to standard protocols. After transfer to HybondC+ nitrocellulose membranes (Amersham), MICL was detected by probing with HB3, followed by a goat anti-mouse HRP, and exposure with the ECL-plus kit (Amersham). To de-N-glycosylate cell lysates, equal quantities of protein were incubated with PNGase F (Roche), in sodium phosphate buffer at 37°C for 24 h, prior to running on SDS-PAGE gels.
Immunocytochemistry, for testing HB3 specificity, was performed on fixed and permeabilized transfected NIH3T3 cells, plated in 8-well chamberslides, according to conventional protocols. HB3 staining was detected using an alkaline-phosphatase-conjugated secondary antibody and BCIP/NBT (Vector Laboratories). Cells were counterstained with Nuclear Fast Red (Vector Labs). For anti-MICL immunohistochemistry, fresh frozen tissues, a gift from Satish Keshav (University College, London, UK), were sectioned and stained according to standard protocols, with the help and expertise of Elizabeth Darley (Oxford, UK). HB3 staining was detected with a peroxidase-conjugated goat-anti-mouse secondary antibody (Vector laboratories) and diaminobenzidine (DAB). Tissues were counterstained with methyl green (Vector Labs).
We would like to thank Philip Taylor, Elizabeth Darley, Richard Stillion, Luisa Martinez-Pomares, John Davies, Martin Stacey, Satish Keshav, Joanna Miller, Elizabeth Soilleux, Eamon McGreal, Clare Jolly and Brenda Jones (Institute of Animal Health), for reagents and advice. Anthony Segal and Daniel Marks are thanked for introducing us to the skin window technique. We gratefully acknowledge a Theodore Williams’ Scholarship for ASJM and funding from the Wellcome Trust, Medical Research Council (SA) and the Edward Jenner Institute for Vaccine Research. GDB is a Wellcome Trust International Research Fellow in Biomedical Science in South Africa. “MICL” is pronounced ‘mickle’, as in the nonsensical proverb: “Monae a mickle maketh a muckle”.