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Uncorrected Version. Published on April 22, 2009 as DOI:10.1189/jlb.1108682

Article

Cellular expression and antimicrobial function of a phylogenetically conserved novel histone 1x-like protein on mouse cells: a potential new class of pattern recognition receptor Donald L. Evans,* Meghan A. Connor,* Lauren D. Moss,* Sarah Lackay,† John H. Leary III,* Thomas Krunkosky,‡ and Liliana Jaso-Friedmann*,1 Departments of *Infectious Diseases, †Pathology, and ‡Anatomy and Radiology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA RECEIVED NOVEMBER 7, 2008; REVISED JANUARY 30, 2009; ACCEPTED FEBRUARY 24, 2009. DOI: 10.1189/JLB.1108682

ABSTRACT A H1x-like protein (i.e., NCAMP-1) is expressed on the membrane and in GEs from fish NK-like cells. In the present study, we identify the imprinting control region mouse NCAMP-1 ortholog using NCAMP-1 polyclonal antibodies and mAb. Polychromatic flow cytometry revealed NCAMP-1 expression on PBLs (Gr-1⫹ PMNs were 21.1% NCAMP-1⫹; DX-5⫹ NK cells were 12.2% NCAMP-1⫹), mesenteric LN cells (CD11c⫹ DCs were 23.2% NCAMP-1⫹; Gr-1⫹ PMNs were 24.8% NCAMP-1⫹; CD21⫹ B cells were 17.8% NCAMP-1⫹), and splenocytes (CD11c⫹ were 39.6% NCAMP-1⫹; Gr-1⫹ PMNs were 40.9% NCAMP-1⫹; DX-5⫹ NK cells were 24.3% NCAMP-1⫹; CD21⫹ B cells were 28.5% NCAMP-1⫹). Western blot analysis using pNCAMP-1 and GEs from RAW 264.7 cells produced a 32-kDa signal. GEs from RAW 264.7 cells produced a significant reduction in Escherichia coli CFU. This antimicrobial killing activity was inhibited by pretreatment of the extract with (polyclonal) anti-NCAMP-1. Treatment with preimmune serum did not reduce bacterial cell killing. Confocal microscopy using NCAMP-1 and LAMP-1 mAb demonstrated that NCAMP-1 was located on the membrane and in cytosolic vesicles of RAW 264.7 cells and did not appear to colocalize with LAMP-1. NCAMP-1 may participate as a bifunctional

Abbreviations: AMP⫽antimicrobial peptide(s), BBS⫽borate-buffered saline, BBSNS⫽BBS with 0.1% saponin, 0.1% NaN3, BM⫽bone marrow, DAPI⫽4⬘,6-diamidino-2-phenylindole, DCs⫽dendritic cells, DP⫽doublepositive, FCS⫽forward-scatter, GE⫽granule extract, H1x⫽histone 1x, IC⫽isotype control, LAMP-1⫽lysosome-associated membrane protein 1, LN⫽lymph node(s), MH⫽Mueller-Hinton, NCAMP-1⫽NCC cationic antimicrobial protein-1, NCC⫽nonspecific cytotoxic cells, NET⫽neutrophil extracellular traps, NrbIgG⫽normal rabbit IgG, PAB⫽PBS, 0.1% sodium azide, 0.1% BSA, PBSN⫽PBS with 0.1% NaN3, PBSNS⫽PBSN with 0.1% saponin, PMNs⫽polymorphonuclear neutrophils, pNCAMP-1⫽polyclonal antiNCAMP-1, s⫽soluble, SP⫽single-positive, SSC, side-scatter

0741-5400/09/0086-0001 © Society for Leukocyte Biology

protein on cells. It is expressed on the membranes of phagocytic cells, NK cells, and APCs in mice as well as in the granules of macrophages. In phagocytic cells, NCAMP-1 may participate in a nonregulated exocytosis pathway of cellular secretion. J. Leukoc. Biol. 86: 000 – 000; 2009.

Introduction The functions and structures of several hundred different AMP and proteins have been described previously [1–5]. Their roles in innate as well as adaptive immune responses continue to be advanced. The unique and varied chemistries and phylogenetic occurrences of antimicrobials from plants, invertebrate, and vertebrate species (indirectly) attest to the relevance of antimicrobials in maintenance of plant and animal health. One of the least-characterized classes of AMP are those belonging to linker and core histones, which must now be considered as actively mobile molecules within a cell, based on their multifunctional activities. Histones bind to and stabilize nuclear chromatin; histones are secreted by epithelial cells; and histones are released during degranulation of leukocytes and kill bacteria following direct contact. Also, histones obtained from multiple cell types have potent antibacterial activities [6], and they have been identified from diverse terrestrial and aquatic animal species. Among the animal models used in the identification of histone functions, studies using teleosts are important examples of the relevance of these proteins in innate immune activities. Histone-like proteins and peptides with antimicrobial activity have been isolated from salmon blood, liver, intestine, skin, and mucus [7–9]. Catfish skin, epithelial cells, and mucus con1. Correspondence: Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. E-mail: [email protected]

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tain H2a-like (Parasin-I) and H2b-like molecules [10 –12]. We have recently identified [13] a protein from catfish NCC referred to as NCAMP-1. This protein is expressed on the membrane of NCC (the teleost equivalent of mammalian NK cells [14 –17]) and as such, binds single-base oligodeoxyguanosine, GpC, and CpG. Interestingly, the complete amino acid sequence of NCAMP-1 (Accession Numbers AAQ99138 and AY324398) revealed 42.4% and 51.2% identity with human and zebrafish (respectively) histone family member H1x. Furthermore, NCAMP-1 is a membrane protein that binds CpG in a human NK cell line, YT-INDY [18]. Literature relevant to the present study addresses molecular mechanisms of granule composition and membrane expression of H1x-like NCAMP-1 in/on mouse cells. Linker and core histones were shown to be expressed previously by the membranes of mouse cells [19 –22]. One function may be the participation in receptor-mediated endocytosis. In one study [23], thyroglobulin-cross-linked H1 receptors on mouse macrophages resulted in subsequent localization into endocytic vesicles. Further evidence [24] for the extranuclear expression of histones was that histone H1 was found in endocytic vesicles of mouse J774.1 and RAW 264.7 macrophages and in resident peritoneal macrophages of mice. GEs from these cells exhibited antimicrobial activity against Gram-negative and Grampositive bacteria. In other studies [25, 26], relatively large concentrations of H1 were found in association with human and pig intestinal epithelial cells. These studies indicated that histones (especially linker histones) were associated with plasma membrane expression during the afferent phase of the innate immune response. However, other data clearly demonstrated that histones also are found in indirect association with the membranes of apoptotic cells and in NET [27–29]. Examples exist for the expression of histones on viable cells [18, 19]; histones and nucleosomes were found in cell lysates from activated lymphoblasts [30]; and H1 has been found in the extracellular matrix of regenerating skeletal muscle cells [31]. These data indicated clearly that linker and core histones may associate nonspecifically with cell membranes by noncovalent ionic interactions rather than by specific receptor-ligand interactions. These data are supported by findings that linker and core histones cross plasma membranes directly (outside to inside), independent of endocytosis but dependent on electric charge differences. This process resulted in direct translocation of histones into the cytosol [32]. We predict that NCAMP-1/H1x is processed similar to other membrane proteins, and expression proceeds in the opposite direction (e.g., from the nucleus to the endoplasmic reticulum and on to the cell membrane). This is supported by data [33] showing that not only is H1x transcription replication-independent, but the H1x gene contains two tandem polyadenylation sites. Additionally, the human H1x gene maps to a different chromosome compared with the main histone gene cluster; there is only 20 –30% similarity between H1x and the other known H1 subtypes, and the two amino acids that are considered to be responsible for high-affinity binding of H1 to chromatin in vivo are not conserved in H1x [33]. There

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is a high possibility that NCAMP-1 may be a new member of the H1x family of histones that participate in innate immunity. In the present study, we address these issues in a mouse model. A novel H1x-like membrane protein (e.g., NCAMP-1) is identified on mouse cells using a NCAMP-1 mAb. Polychromatic flow experiments indicated cell subset expression of NCAMP-1. Possible functions in innate immunity were demonstrated by finding antimicrobial activities of NCAMP-1 from macrophage granules, and confocal analysis demonstrated that NCAMP-1 may be associated with a secretory exocytosis pathway rather than a constitutive component of a secondary granule/phagolysosome endocytic pathway.

MATERIALS AND METHODS

Animals and cells In vivo-derived macrophages were obtained from outbred imprinting control region-hydroxysteroid dehydrogenase mice (Harlan Laboratories, Indianapolis, IN, USA). Three- and 9-month-old mice were used (differences in phenotype analysis were not age-related; data not shown). The in vitro-cultured cell line was RAW 264.7 (ATCC TIB 71).

Reagents and antibodies mAb were obtained from eBioscience (San Diego, CA, USA): PE-Cy5 antimouse MHC Class II (Cat. #15-5321) used 0.015 ␮g/105 cells; PE-Cy7 antimouse CD11b (Cat. #25-0112) used 0.125 ␮g/105 cells; allophycocyanin anti-mouse CD45.1 (Cat. #17-0453) used 0.5 ␮g/105 cells; PE anti-mouse CD49b (Cat. #12-5971) used 0.5 ␮g/105 cells; PE anti-mouse CD21/CD35 (Cat. #12-0211) used 0.25 ␮g/105 cells; PE-Cy5.5 anti-mouse Ly-6G (Cat. #35-5931) used 0.03 ␮g/105 cells; PE anti-mouse CD3 (Cat. #12-0031) used 0.5 ␮g/105 cells. mAb were obtained from BD PharMingen (San Diego, CA, USA): biotin-conjugated hamster anti-mouse CD11c (Cat. #553800) used 0.5 ␮g/105 cells; streptavidin allophycocyanin-Cy7 (Cat. #554063) used 0.2 ␮g/105 cells. mAb generated in the lab: M2 (IgM isotype control used 5 ␮g/105 cells); IgM anti-NCAMP-1 mAb 9C9 [17] used 5 ␮g/105 cells.

Cell preparations and phenotyping Single cell suspensions from spleen and LN were obtained by physical disruption (mesh screen disruption/spleen and glass-slide disruption/LN). Cells were resuspended in 15 ml RPMI 1640 and centrifuged (900 g). Pellet (4°C) was resuspended in 10 ml RBC lysing solution (154 mM ammonium chloride, 10 mM potassium bicarbonate, 0.082 mM EDTA) for 30 s, and 5 ml 2⫻ PBS (pH 7.4) was added. Cells were counted in a hemacytometer, and 2.5 ⫻ 105 cells were dispensed, centrifuged (900 g), and resuspended in 50 ␮l PAB (Sigma A2153-10G, Sigma Chemical Co., St. Louis, MO, USA) at 4°C. mAb were added (60 min/4°C), washed, and resuspended in PAB for flow analysis for direct-conjugated mAb, or anti-IgM-FITC conjugate (1:50 in PAB; Sigma F-9259) was added (30 min/4°C) for in-house mAb. Blood was obtained by cardiac puncture, and RBC were removed by centrifugation (700 g/30 min/20°C) over 3 ml Histopaque 1077 (Sigma #10771). Cells were removed from the 1077 cushion, washed, and counted, and 1 ⫻ 105 cells were added to 12 ⫻ 75 tubes. Cells were resuspended in 100 ␮l PAB (4°C), and mAb were added sequentially at concentrations indicated previously. After 1 h/4°C, cells were washed in PAB (4°C) and analyzed by polychromatic flow cytometry. Flow analysis [BD FACSCanto™ II system (BD Biosciences, San Jose, CA, USA) with a three-laser eight-color system (405, 488, and 633 nm)] for polychromatic marker combinations consisted of the following mAb: for B cell identification, CD45/MHCII/CD21/CD11b/NCAMP-1 [17]; macrophages and DC identification, CD45/MHCII/CD11c/CD11b/NCAMP-1; T cell identification, CD3/NCAMP-1; NK cell identification, DX5/NCAMP-1;

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Evans et al. Histone 1x-like pattern recognition receptor and neutrophil identification, Gr-1/NCAMP-1. Results are representative of four independent experiments.

Western blot GEs, prepared from RAW 264.7 cells (see Granule purification below), were run on 11% SDS gels (reducing) and transferred to nitrocellulose. Filters were blocked for 15 min at room temperature (Superblock, Pierce, Rockford, IL, USA, #37545, with 0.05% Tween 20). Rabbit pNCAMP-1 was prepared in-house. Protein A-purified NRbIgG was used as isotype control. Primary antibody dilutions were made in BBS (pH 8.5) supplemented with BSA (1%) and Tween 20 (0.05%). Peroxidase-conjugated goat anti-RbIgG secondary antibody (Pierce, #31463, 1:50,000 dilution) was diluted in Superblock blocking buffer. Blots were developed with ECL (Pierce Supersignal West Pico #1856135).

(LAMP-1, Alexa Fluor 488-conjugated, eBioscience; 0.5 ␮g/sample) was added and incubated for 30 min followed by four washes in PBSNS. Slides were mounted with Prolong Gold anti-fade with DAPI (Invitrogen). Samples were cured overnight and then sealed and stored at 4ºC pending scanning. Images were acquired with a Zeiss LSM 510 laser-scanning system in Multitrack mode, and stacks were assembled using the Zeiss LSM Image browser. Images were manipulated further with Adobe Photoshop.

Statistical analysis One-way ANOVA with Tukey’s post-test was used to analyze data where indicated (GraphPad Prism 5 software, GraphPad Software, San Diego, CA, USA). P values ⬍0.05% were considered significant.

Granule purification

RESULTS

RAW 264.7 cells were harvested, counted, and resuspended in PBS. Cells were pelleted at 900 g for 5 min and resuspended on ice for 10 min in dounce buffer (10 mM TrisCl, pH⫽7.6, 0.5 mM MgCl2) containing 1 ␮g/ml each of four protease inhibitors: aprotinin (Sigma Chemical Co., A1153), pepstatin (Sigma Chemical Co., P4265), leupeptin (Sigma Chemical Co., L2023), and PMSF (Sigma Chemical Co., P7626). Cells were dounce-homogenized for 30 strokes followed by addition of 2.5 ml tonicity restoration buffer (10 mM TrisCl, pH⫽7.6, 0.5 mM MgCl2, 0.6 M NaCl) plus protease inhibitors. Cells were centrifuged at 400 g for 5 min at 4°C to remove nuclei. The supernatant was removed and spun at 27,000 g for 20 min at 4°C. The pellet was resuspended in 5% acetic acid and sonicated three times for 5 s each. The tube was place on a rocker at 4°C for 24 h followed by centrifugation at 27,000 g for 20 min at 4°C. The supernatant was frozen into aliquots and vacuum-centrifuged to remove acetic acid. Dried extracts were resuspended in water and the drying process repeated. The concentrated GE protein was resuspended in a 10-mM sodium phosphate buffer, pH⫽8.0, and stored at –20°C.

Expression of NCAMP-1 on mouse leukocytes by phenotype analysis

RAW GE killing assay Purified RAW GE (0.125 ␮g/␮l) was incubated with 35 ug rabbit preimmune serum or anti-NCAMP polyclonal antibody in 10 mM sodium phosphate buffer (pH⫽8.0) for 30 min. Polymyxin b (1 ␮g/mL) was used as a positive control. A bacterial culture of Escherichia coli APEC 3751 was grown in MH broth and diluted to 105 CFU/mL. A relationship between absorbance at 600 nm and CFUs was determined previously in our laboratory. Fifty microliters (5000 CFU) was added to an equal volume of GE or GE preincubated (30 min, room temperature) with anti-NCAMP-1 polyclonal antibody or NRbIgG. Phosphate buffer (10 mM, pH 8) was used to dilute GE and antibodies to the desired concentrations and final assay volume (100 ul). Assay tubes were placed in an incubator at 37°C for 2 h. Serial dilutions were made, and the bacteria were plated on MH agar plates and placed in a 37°C incubator overnight. CFUs were counted after 24 h. Results are expressed as CFU/ml.

Confocal microscopy Raw 264.7 cells were seeded onto eight-well chamber slides (Lab-Tek II, Nunc, Rochester, NY, USA) and grown overnight at 37°C. All subsequent steps were done at room temperature unless noted otherwise. Slides were washed 2⫻ in PBSN followed by fixation/permeablization with 4% paraformaldehyde in PBSNS for 20 min. Slides were washed 2 ⫻ 5 min (PBSNS). Cells were incubated in an Image-iT signal enhancer (Invitrogen, Carlsbad, CA, US) for 30 min and washed 1⫻ with PBSNS, followed by 2⫻ washes with BBSNS (pH 8.5). Samples were blocked with 10% normal goat serum in BBSNS for 15 min. Rabbit anti-NCAMP-1 or NRbIgG in BBSNS was added and incubated for 1 h. After washing (2⫻5 min in BBSNS; 2⫻5 min in PBSNS), goat anti-RbIgG Texas Red (Invitrogen) was added (1 h in PBSNS). Slides were washed (4⫻5 min; PBSNS). Anti-mouse CD107a

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Polychromatic flow cytometry was conducted using mouse PBLs, splenocytes, LN, and BM cell preparations. Figure 1, A and B, is the polychromatic analysis of NCAMP-1 expressed on PBLs detected with mAb 9C9. Granulocytes, NK cells, and monocytes expressed the highest levels of NCAMP-1. T cells were essentially negative. Polychromatic analysis of NCAMP-1 expression by splenic (Fig. 2) and LN (Fig. 3) subsets indicated that granulocytes, DCs, and B cells expressed the highest percentage positive populations. Figure 4 is the staining of BM cells with the gating strategy and mAb panel as described previously. The lymphoid subsets stained higher for NCAMP-1 than the myeloid cells. A summary of the characteristics of each tissue staining is given in Table 1.

NCAMP-1 is present in GEs and is a membrane protein Figure 5A is a Western blot of purified RAW 264.7 GE probed with pNCAMP-1. Lane 1 is pNCAMP-1, lane 2 is preimmune rabbit serum from the NCAMP-1-immunized animal, and lane 3 is the total protein stain of the GE. Two bands were detected at 31–32 kDa. Flow cytometry analysis (Fig. 5B) confirmed the expression of NCAMP-1 on RAW 264.7 membranes using mAb anti-NCAMP-1. Moreover, results from three independent experiments confirmed that binding of mAb 9C9 to RAW 264.7 competed with the binding of FITC-labeled CpG in a dosedependent manner. Inhibition of FITC-labeled CpG binding ranged between 35% and 60% (data not shown). Similar results have been described previously for catfish NCC and human YT-INDY cells [13, 18].

Antimicrobial killing activity of NCAMP-1 obtained from RAW 264.7 granules Granules were purified from RAW 264.7, and the extract was tested against E. coli for antimicrobial activity. Figure 6 shows that the RAW 264.7 extract (RAW GE) as well as the polymyxin b-positive controls produced almost 100% inhibition of growth of the bacteria. Treatment of E. coli with the pNCAMP-1 or NRbIgG (only) had no effect on growth. Pretreatment of the RAW 264.7 GE with anti-NCAMP-1 polyclonal antibody removed

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that an abundant LAMP-1 signal was observed in these cells. NCAMP-1 was observed in cytosolic “vesicles” and along the margin of RAW 264.7 membranes (Fig. 7, B and C). The two fluorescent signals, however, did not colocalize.

Figure 1. Polychromatic analysis of PBL expression of NCAMP-1. (A) MHC-II and CD45 DP cells were analyzed for CD11c and CD21 expression of NCAMP-1. (B) GR-1, CD3, and DX5 SP cells were analyzed for expression of NCAMP-1.

the bacterial killing activity of the GE compared with NRbIgG (P⬍0.05).

Analysis of NCAMP-1 and LAMP-1 localization in RAW 264.7 cells To determine the cytoplasmic localization of NCAMP-1 relative to endocytic vesicles in phagocytic cells, RAW 264.7 cells were fixed, permeabilized, and labeled with anti-LAMP-1 and antiNCAMP-1 mAb (Fig. 7). Confocal microscopy analysis revealed

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Figure 2. Polychromatic flow analysis of spleen cells for expression of NCAMP-1. Total cell populations indicated by P1–P3. Arrows indicated gating strategies. (A) MHC-II and CD45 DP cells were gated on P3 and NCAMP-1, subsequently analyzed for expression on CD11b and CD11c SP cells. Gr-1 cells expressing NCAMP-1 were gated on P2. (B) Arrows show the P1 gating strategies for NCAMP-1 expression on MHC-II/CD45 and CD11b/CD21 DP cells (28.5%). Cells gated on P2 were SP CD3 and SP DX5. Percentages of NCAMP-1-positive cells were of the immediate preceding parent population.

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Evans et al. Histone 1x-like pattern recognition receptor

Figure 4. Polychromatic analysis of BM cells for NCAMP-1 expression. (A) Gated total cell populations of BM cells are shown as P1 and P2, and P2 cells were stained with CD11b, Gr-1, and CD11c, and NCAMP1-positive cells within each of these parent populations are shown. (B) P1- and P2-gated cells were analyzed for CD21, CD3, and DX5, and NCAMP-1-positive cells within each of these parent populations are shown. FS, FSC.

DISCUSSION Histone proteins that manifest activities other than linker and core functions and that appear in diverse cellular and extracellular locations have been reported [6]. The cytotoxic activity

TABLE 1. Comparisons of NCAMP-1 Expression on Mouse PBL, LN, Splenocytes, and BM Figure 3. Polychromatic analysis of LN cells for expression of NCAMP-1. (A) Separation of LN cells by size and scatter properties and P1–P3 gating strategy. (B) P2 and P3 DP (MHC-II/CD45) cells were stained with CD11b, CD11c, or Gr-1, and SP cells were analyzed for expression of NCAMP-1. (C) Cells from P2, which were CD11b/CD21 DP, expressed 17.8% NCAMP-1; CD3-positive cells from the P3 parent were negative for NCAMP-1; and DX5-positive P3 cells were 9.5% membrane NCAMP-1-positive. Cells were not gated on MHC-II/CD45.

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Percentage positive NCAMP-1

Macrophages (CD11b) Granulocytes (Gr-1) Dendritic cells (CD11c) B cells (CD21) T cells (CD3) NK cells (DX5)

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PBL

LN

SPLEEN

BM

— 21.1 6.4 8.2 neg 12.2

9.9 24.8 23.2 17.8 neg 9.5

4.1 40.9 39.6 28.5 neg 24.3

8.0 9.6 7.6 96.7 27.9 28.9

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(azurophilic), secondary, and gelatinase granules [37]. More direct evidence linking the presence of H1 and antimicrobial activities was demonstrated by finding relatively large concentrations of H1 in association with human and pig epithelial cells [25, 26]. We have shown previously [13, 17] that rNCAMP-1 has antimicrobial activity against Gram-negative and Gram-positive bacteria, and granule-containing extracts from NCC contain NCAMP-1, which has antimicrobial activity. In the present study, NCAMP-1, a H1x-like protein reported previously [13] to be expressed on the membrane and in GEs of NCC of catfish [17], was found not only in the granules of RAW 264.7 mouse macrophages but also on the outer membrane (Figs. 5 and 7) of these cells. Confocal examination of RAW 264.7 cells using anti-NCAMP-1 and anti-LAMP-1 mAb (Fig. 7) revealed that although NCAMP-1 was observed in cytosolic vesicles of these cells, these two proteins did not colocalize to the same vesicle. These data indicated that NCAMP-1 may be associated with storage secretory vesicles that function within a constitutive exocytosis pathway. Additional evidence for a possible role of NCAMP-1 mobilization and secretion by phagocytic cells during innate immune responses was shown in Figure 6, where RAW 264.7 was shown to contain cytosolic NCAMP-1, which may be available for secretion. The relatively high bacterial killing activity of granule-containing acid extracts from RAW 264.7 cells was inhibited specifically by pNCAMP-1. Our data suggest that because of a lack of colocalization of NCAMP-1 with LAMP-1 in mouse macrophages, NCAMP-1 may participate in a stimulus-induced secretion model similar to

Figure 5. RAW 264.7 cells express membrane NCAMP-1, and GEs contain NCAMP-1. Western blot analysis of the purified granules from RAW 264.7 cells is shown (A). GEs were prepared as described previously. Lanes 1 and 2 are rabbit prNCAMP-1 and preimmune serum, respectively, and lane 3 is the total protein stain of RAW 264.7 GE. (B) Staining of nonpermeabilized RAW 264.7 with mAb anti-NCAMP-1. Shaded histogram is the isotype control. FL1, Fluorescence 1.

of histone (H1) for eukaryotic tumor cells has been described [34]. That this nuclear “dwelling” protein could also be involved in antibacterial functions was suggested by others, demonstrating the presence of H1 in cytoplasmic fractions from PMNs [35]. Subsequently, many excellent reviews [36] have shown that linker and core histones are participants in potentially important antimicrobial functions. The role of histones (especially H1) in antimicrobial killing during innate immune responses was suggested indirectly by proteomic analysis of PMN granule contents. H1 family members, H2B and H4, were found in the PMN granule contents of human primary

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Figure 6. Antimicrobial activity of RAW 264.7 GE and its depletion with NCAMP-1-specific polyclonal antibody. E. coli (5000 CFU) was added to an equal volume of the indicated test mixtures (after a 30min preincubation). The following amounts were used (per test): polymyxin b, 1 ug; anti-NCAMP-1 and NRbIgG, 10 ug each; RAW GE, 100 ng. Samples were run in quadruplicate. Data shown are the mean ⫾ sem and are representative of four independent experiments. One-way ANOVA with Tukey’s post-test (Graphpad Prism 5) was used to evaluate significance.

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Figure 7. Localization of NCAMP-1 and LAMP-1 in RAW 264.7 cells, which were permeabilized and fixed as described previously and examined by confocal microscopy. (A) Panel 1, LAMP-1 only; Panel 2, DAPI only; Panel 3, IC instead of anti-NCAMP-1; and Panel 4, IC ⫹ anti-LAMP-1 ⫹ DAPI. (B and C) Same as A, except Panels 3 and 4 contain anti-NCAMP-1 instead of IC.

secretory lysosomes of cytotoxic T cells and NK cells (which are LAMP-3⫹, LAMP-1–, LAMP-2–), or localization of cytosolic NCAMP-1 may be analogous to early/sorting endosomes of

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macrophages or azurophilic storage granules of PMNs (which are also LAMP-1–) [37]. These assumptions would place NCAMP-1 as a major participant in a secretion model rather than an intracellular antimicrobial constituent of late endosomes and phagolysosomes. It was of interest that two different molecular weight species of NCAMP-1 were observed by Western blot analysis (Fig. 5) of RAW 264.7 supernatants and that these molecular weight forms were slightly different than that for teleost NCAMP-1 [13]. This microheterogeneity of molecular weight has also been observed in the human NK-like cell line YT-INDY (data not shown). It is possible that in mammals, the phylogenetically conserved, nascent 31- to 32-kDa low molecular weight form of NCAMP-1 observed in GEs (Fig. 5) and on teleost membranes resulted not from multiple genes or alternative RNA processing but from processing of NCAMP-1 in the secretory vesicle from the 32-kDa native protein. Proteome analysis of neutrophil granule subsets (gelatinase, specific and azurophilic granules) indicated that certain histone H1 family members, H2B and H4, were among the 286 proteins identified [37]. A novel finding in the present study was that NCAMP-1 was localized not only in (putative) secretory vesicles but also was expressed on the membranes of different myeloid and lymphoid lineages of cells. We demonstrated in Figures 1– 4 that in general, APCs and NK cells, regardless of the tissue source, expressed relatively high levels of membrane NCAMP-1. Additionally, CD11c⫹ DCs from the spleen had high levels of expression. T cells and CD11b⫹ cells were negative or had low NCAMP-1 binding. At present, the relevance of variations in expression levels among different leukocyte classes and macrophages is not known. It is possible that the molecular similarity between NCAMP-1 and linker histone proteins might suggest a paradigm shift regarding the role of H1-like proteins in immune responses. That is, a new H1 subtype may exist regarding histone function. This new isoform (e.g., NCAMP-1) may be dedicated to perform functions of innate immunity rather than chromosome binding and stabilization. The present study indicated that classification of H1 proteins may become an important issue relevant to studies of H1x and H1. There are 10 H1 proteins in mammals [33], and little is known about the biological functions of H1x. The most relevant H1 subtype for analysis of the identity and function of H1x is H1°. Expression of the H1° subtype is independent of whether a cell is undergoing replication; it is expressed in terminally differentiated cells (e.g., PMNs), and it contains two polyadenylation sites at the 3⬘-untranslated region [38]. H1x mRNA is also polyadenylated [33], and similar to H1°, expression is replication-independent, and the H1x gene has been mapped to a different human chromosome compared with the other H1 subtypes [39]. All of these data perhaps suggest that new functions must exist for the H1 subtype family including a new member, NCAMP-1. Our hypothesis, which predicts that different subtypes of the H1 family have highly diversified cellular and nuclear functions, is supported by recent studies of the role of H1 in the killing of bacteria as a component of NET [40 – 42]. The principal component of the NET is H1-linked to chromatin frag-

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ments that have become reflected to the exterior of the PMN. The mechanism of cellular release from PMNs of H1 in context with chromatin fragments has only been suggested to be associated with a unique pathway of cell death. The mechanism of formation of NETs is apparently not dependent on apoptosis or necrosis. The executioner caspases (caspase-3, -6, -7) are not converted in the dying PMN (e.g., the cell is not undergoing apoptosis), and the nuclear envelope of the PMN appears to disintegrate early in the death process (an early sign of apoptosis but not necrosis) [40 – 42]. The antimicrobial activity of NET is linked with the H1 protein in association with the extracellular chromatin fragments of the NET. A second type of H1 function associated with dead/dying cells has been shown to occur regarding the adhesion of the 987P fimbriae of enterotoxigenic E. coli to H1 released from dead epithelial cells [26]. This complex then becomes bound to the brush border of mucosal cells. This second stage of binding was thought to be dependent on ionic charge interactions between negatively charged brush border and positively charged H1. The relevance of this type of interaction in the intestine regarding bacterial pathogenesis or activation of innate immunity in the enterocytes remains to be determined. A fourth major function (in addition to NCAMP-1 as a pattern recognition protein, chromatin stabilization, and NET antimicrobial activity) of the H1 family must now be considered to include the participation of H1 as an effective adjuvant in vaccine formulations [43]. H1 isolated from Leishmania major was expressed in recombinant form and used in a vaccine experiment for protection of vervet monkeys, who when immunized with the rH1 protein, had reduced development of lesion size compared with controls. Functions from chromosome stabilization to participation in the final dying act of a PMN or intestinal epithelial cell must now place this important group of proteins as phylogenetically significant effectors of animal survival. We have shown that NCAMP-1 is widely expressed by many myeloid and lymphoid cells and is found in abundant concentrations in macrophage granules. It is possible that as an active antimicrobial, NCAMP-1 may be secreted from phagocytic cells, whereas as a membrane protein (Figs. 1–5), NCAMP-1 is positioned to function as a pattern recognition receptor. There is ample evidence for the existence of membrane protein receptors that also exist in soluble form. sIL-2R1 [44], sTNF-R [45], and sIL-6R-␣ [46] are examples of membrane receptors that also have biological function as secreted proteins. Thus, NCAMP-1 is phylogenetically conserved from fish to mice and as such, may serve multiple capacities: pattern recognition receptor, secretion by exocytosis, and bactericidal activity in soluble form.

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KEY WORDS: innate immune antibacterial peptides• 䡠 host-pathogen interactions

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