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SIRPα/CD172a and FHOD1 Are Unique Markers of Littoral Cells, a Recently Evolved Major Cell Population of of Human This information is current as of September 28, 2021. Javier Gordon Ogembo, Danny A. Milner, Jr., Keith G. Mansfield, Scott J. Rodig, George F. Murphy, Jeffery L. Kutok, Geraldine S. Pinkus and Joyce D. Fingeroth J Immunol 2012; 188:4496-4505; Prepublished online 4 April 2012; Downloaded from doi: 10.4049/jimmunol.1103086 http://www.jimmunol.org/content/188/9/4496 http://www.jimmunol.org/ References This article cites 95 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/188/9/4496.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

SIRPa/CD172a and FHOD1 Are Unique Markers of Littoral Cells, a Recently Evolved Major Cell Population of Red Pulp of Human Spleen

Javier Gordon Ogembo,*,†,1 Danny A. Milner, Jr.,†,‡,1 Keith G. Mansfield,†,x Scott J. Rodig,†,‡ George F. Murphy,†,‡ Jeffery L. Kutok,‡ Geraldine S. Pinkus,†,‡ and Joyce D. Fingeroth*,†

Asplenic individuals are compromised not only in their ability to destroy infectious agents, but are at increased risk for death from autoimmune disease, certain tumors, and ischemic disease. Enhanced mortality is attributed to lack of sequestered in spleen that efficiently engulf and destroy appropriate targets, although related cells are found elsewhere. To determine whether a unique population regulates RBC-pathogen clearance and filtration of altered self, we reviewed the anatomic literature and an-

alyzed in situ by immunohistochemistry and immunofluorescence the expression patterns of a little-characterized cell that dom- Downloaded from inates the splenic red pulp of humans and closely related primates: the venous sinus-lining or littoral cell (LC). High expression of the formin domain protein 1 outlines the LC population. Although LCs are endothelial-like in distribution, they express several -directed proteins, the RBC Duffy Ag receptor for chemokines and coreceptor CD8a/a, yet they lack lineage-associated markers CD34 and CD45. Strikingly, SIRPa (CD172a) expression in human spleen concentrates on LCs, consistent with recent demonstration of a key role in RBC turnover and elimination versus release of infected or altered self. + + + 2 2 Our results indicate human LCs (SIRPa , formin homology domain protein 1 , CD8a/a , CD34 , CD45 ) comprise a highly http://www.jimmunol.org/ plastic barrier cell population that emerged late in primate evolution coordinate with CD8 expression. Unique to Hominidae, LCs may be the ultimate determinant of which cells recirculate after passage through human spleen. The Journal of Immunology, 2012, 188: 4496–4505.

ndividuals who are asplenic or functionally asplenic (hypo- a filter that mediates the ultimate retention and destruction of splenic) are unable to eliminate many bacterial and parasite senescent and modified cells (5, 6). pathogens (1). They also display an increased risk for auto- Opsonization with Ab and/or complement enhances clearance of

I by guest on September 28, 2021 immune disease (1), some (2), and ischemic cardiac dis- foreign Ags, cell debris, and altered self by phagocytes (1, 7). In ease (2, 3). Perhaps this is not surprising because the spleen is the humans, inflammatory particles that are opsonized and immobi- largest secondary in . However, lized by complement fragments are regularly transported through although the overall organization of splenic is similar the circulation tethered by type 1 (CD35) on to that of lymph nodes, the spleen is not connected to the lym- the RBC membrane for efficient phagocytosis at distant locations phatic system (4). Rather, soluble and particulate Ags, pathogens, (4, 8, 9). This process, a sophisticated mechanism known as im- RBCs, and altered, apoptotic, necrotic, and tumor cells are all mune adherence clearance, evolved with mammals to remove delivered to and leave the spleen via the circulation. In contrast inflammation-inducing particles from (10). However, utili- with white pulp, the red pulp of mammalian spleen serves as zation of RBCs for immune adherence clearance is a recent de- velopment in the evolution of the immune system restricted to humans and closely related primates, because nonprimates, in- *Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, MA 02215; †Harvard Medical School, Boston, MA 02115; ‡Department of , cluding rodents, primarily rely on -based transport mech- Brigham and Women’s Hospital, Boston, MA 02115; and xDivision of Primate Med- anisms (11, 12). Much evidence indicates and spleen are icine, New England Primate Research Center, Southborough, MA 01772 where CD35-bound immune complexes (ICs) are delivered (9), 1 J.G.O. and D.A.M. contributed equally to this work. although precisely how ICs are removed at each location is not Received for publication October 26, 2011. Accepted for publication February 29, well understood. 2012. RBCs themselves (residual IC loaded, infected, and aging) are This work was supported by National Institutes of Health Grants R01AI063571 (to sequestered, variably internalized, and removed as they course J.D.F.) and K23AI072033 (to D.A.M.), an American Heart Association Grant-in-Aid (to J.D.F.), and a fellowship from the Research Institute (to J.G.O.). through the red pulp of the spleen (13). Precisely where and how J.D.F., J.G.O., and D.A.M. designed and analyzed all experiments, reviewed the RBCs and other altered cell types delivered by the circulation are literature, and wrote the manuscript. J.G.O. and D.A.M. performed most experiments. destroyed or filtered is unknown. The stroma of human red pulp is K.G.M., S.J.R., G.S.P., G.F.M., and J.L.K. provided expertise and additional samples composed of the and the sinusoids (14). In for analysis. contrast with human, the mouse has an asinusal spleen, and thus it Address correspondence and reprint requests to Dr. Joyce D. Fingeroth, Division of Infectious Diseases, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, is architecturally and functionally distinct (4, 15–21). Human red CLS-445, Boston, MA 02215. E-mail address: jfi[email protected] pulp has a high content of diverse phagocytes; however, to return Abbreviations used in this article: DARC, Duffy Ag receptor for chemokines; to the venous circulation, most splenic constituents must pass FHOD1, formin homology domain protein 1; IC, ; IHC, immuno- between or through the sinus-lining cell or littoral cell (LC). Al- histochemistry; LC, littoral cell; LCA, LC angioma. though the morphologic appearance of the LC suggests it may be Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 a key mediator of particle clearance and cellular filtration (13), www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103086 The Journal of Immunology 4497 direct demonstration of function is lacking. In fact, little is known CD47 into the venous circulation may ultimately be determined by about these cells. the LC, a hypothesis examined in this study. Littoral literally means “shoreline” or “tidal,” which accurately describes the polarized cells that line the venous sinusoids of Materials and Methods human spleen with cytoplasmic fronds protruding into the sinus. Tissue LCs comprise ∼30% of the red pulp, and thus are a major con- Normal discarded and unidentified human spleen consented for research stituent of human spleen (22). Morphologically, LCs are elongate was obtained from the Pathology Departments of Beth Israel Deaconess and contain prominent cytoplasmic filaments ( fibers) (23). Medical Center, Brigham and Women’s Hospital, and New England Organ Their abundant cytoplasm is filled with many pinocytic vesicles, Bank. The tissues were obtained in accordance with the policies of the lysosomes, and dense deposits that surround the nucleus. Phago- Institutional Review Board at each of the respective sites. Splenic tissues were processed immediately to optimize conservation of cell morphology cytosed RBCs, leukocytes, hemosiderin, and other debris can be and composition. LCA slides were provided by the Brigham and Women’s visualized within these cells (24–26), even more so in the setting Hospital Pathology Department (to S.J.R., generously provided by Dr. of certain diseases (27, 28). RBCs can also be seen coursing be- Christopher D.M. Fletcher). Archived formalin-fixed, paraffin-embedded, tween adjacent LCs (10, 13). nonhuman primate were obtained from a repository at the New Based on their perisinusal distribution, it was long assumed that England and Southwest Primate Research Centers. Archived formalin- fixed, paraffin-embedded normal mouse (BALB/c) and rat (Sprague Dawley) LCs derive from . However, in 1983–1985, in situ spleen were obtained from the Dana-Farber Cancer Institute. staining showed (22, 29) that LCs also express several Ags found on , as well as CD8, which at that time was believed to Immunohistochemistry of mammalian spleens be T cell restricted. Although CD8 expression was verified (14), Immunohistochemistry (IHC) staining and analysis were performed per Downloaded from the close proximity of LCs to other red pulp components including routine protocol on human, nonhuman primate, and rodent splenic tissues as diverse phagocytes, endothelial cells, and often re- previously described (40). Ab concentrations and reaction conditions were varied as described in Table I. All sections were 5-mm-thick, formalin- sulted in discrepant characterization. Since then work has been fixed, paraffin-embedded splenic tissue sections on individual slides. Two limited. However, the LC angioma (LCA), an unusual splenic independent pathologists assessed reactivity for enumerated Ags. Positive tumor believed to derive from LCs, has been more often described staining of lymphocytes, endothelial cells, and distributed neutrophils and

despite its rarity (24). LCAs are low-grade tumors that contain few served as positive internal controls. Positive staining for LCs http://www.jimmunol.org/ atypical mitoses. Although related by morphology, angioma cells was defined as evidence of peroxidase reaction within the linear and cir- cular LC network either as continuous cytoplasmic or polarized linear appear enlarged, disorganized, and, interestingly, they lack CD8 membrane staining. (24, 30), a consistent marker of normal LCs. A striking clinical observation is frequent association of LCAs with malignancy Detection of CD8 expression on primate spleen by IHC (often lymphoma or solid tumors) at distant sites, mandating in- An IHC protocol for detection of CD8 was performed on primate spleen as vestigation for occult malignancy in any patient diagnosed with previously described (41). In brief, formalin-fixed, paraffin-embedded LCA (31). LCAs have also been associated with several forms of tissues were sectioned at 5 mm, deparaffinized in xylene, rehydrated in sequential ethanol/water washes, and incubated with 3% hydrogen per- autoimmune disease (lupus, inflammatory bowel disease), certain oxide in PBS for 5 min followed by Ag retrieval consisting of microwave hemoglobinopathies (, Punjab), and pretreatment in Trilogy solution (Cell Marque, Rocklin, CA). Dual en- by guest on September 28, 2021 a storage disorder (Gaucher’s). This diversity suggests that rec- dogenous enzyme block (5 min) and protein block (10 min) were used ognition, contact, internalization, and/or filtration of abnormal cell to eliminate nonspecific staining (DakoCytomation, Carpinteria, CA). types or cell products (possibly entosis) (28, 31, 32) drives aber- Sections were then incubated with primary Ab (anti-CD8 clone 1A5; Vector) at 1:50 for 60 min at room temperature followed by incubation rant LC activation and development of the LCA. with Envision Labeled Polymer (DakoCytomation) for 30 min. Ag–Ab We report that formin homology domain protein 1 (FHOD1), complex formation was detected by use of 3,39 diaminobenzidine chro- a member of the formin family of diaphanous-related formins, is mogen (DakoCytomation). An isotype-matched (IgG1) control Ab (mouse highly expressed on human LCs distinguishing them from other IgG1 negative control; catalog no. MCA928; Serotec) was used as irrel- evant control Ab along with standard positive control rhesus tissue. (33). Using high FHOD1 and CD8 expression together with perisinusal localization as an initial guide, we examined, and/ Colocalization of CD8 and CD163 by immunofluorescence or re-examined when discrepant, previously reported patterns of microscopy Ag expression on LCs from normal human spleen. We contrasted Sections of formalin-fixed, paraffin-embedded tissue (6 mM) were prepared this with the LCA, other red pulp inhabitants, rodent, and a spec- and then deparaffinized in xylene twice for 10 min each, followed with trum of primate splenocytes. Endothelial cells that line the venous 2 steps each in 100% ethanol, 95% and 75% ethanol, 3 steps in dH2O, all sinuses of human liver, although similar in distribution, are dis- for 2 min each. The sections were placed in 13 target retrieval solution tinct from LCs that express a unique spectrum of Ags including (DakoCytomation) and boiled in a Pascal pressure chamber (DakoCyto- mation) at 125˚C for 30 s, 90˚C for 10 s, and then allowed to cool down to FHOD1, SIRPa/CD172a, CD8a/a, as well as Duffy Ag receptor room temperature. To evaluate coexpression of CD8 and CD163, primary for chemokines (DARC)/CD234 (34), CD206 (35), stabilin-1 (36), Abs (mouse anti-human CD163 mAb, clone 10D6 [Leica, Buffalo Grove, and other distinctive proteins, despite lacking common lineage- IL] and rabbit anti-human CD8 mAb, clone SP-16 [Abcam, Cambridge, associated markers CD34 (endothelial) and CD45 (hematopoi- MA]) were incubated on the tissue sections overnight at 4˚C. After washing out unbound primary Ab with TBST, the sections were incubated etic). Perhaps most revealing, the unexpectedly high polarized with the appropriate secondary Abs (Alexa Fluor 594 donkey anti-mouse expression of SIRPa on LCs when compared with surrounding red IgG [Invitrogen, Carlsbad, CA] or Alexa Fluor 488 donkey anti-rabbit IgG pulp phagocytes hints at mechanism. SIRPa, a transmembrane [Invitrogen]) at room temperature for 1 h. After washing out unbound protein primarily localized to myeloid cells (37), has been in- secondary Abs, slides were then placed under coverslips with ProLong creasingly implicated as a major regulator of RBC turnover and Gold Anti-Fade containing DAPI (Invitrogen). Sections were analyzed with a BX51/BX52 microscope (Olympus America, Melville, NY). Images innate self-recognition (38, 39). This is achieved through inter- were captured using the CytoVision 3.6 software (Applied Imaging, San action with CD47, a ubiquitous ligand expressed on neighboring Jose, CA). cells that upon engaging SIRPa transduces a negative intracellular signal that blocks phagocytosis. The final decision to destroy or RT-PCR not to release pathogen-bearing and senescent RBCs, circulating RT-PCR to detect FHOD1 message in murine spleen compared with human tumor, and/or other altered cells marked by reduced or absent spleen was performed as described previously (34) using specific (forward 4498 HUMAN SPLENIC LITTORAL CELLS

MG182, reverse MG196) and control (GAPDH: forward MG121, reverse MG122) primers as detailed. Microscopy Visualization of all stained tissues was performed on an Olympus BX41 microscope. Splenic tissue was photographed on an Olympus BX41 mi- croscope using OlympusQColor3 and analyzed with acquisition software QCapture v2.60 (QImaging) and Adobe Photoshop 6.0 (Adobe).

Results Human LCs express a unique spectrum of hybrid Ags In recent years, discovery of many protein markers has greatly refined understanding of the heterogeneity of cells resident in human splenic white pulp; however, little is known about red pulp populations. To begin to uncover the role(s) of the human splenic LC with the goal of determining its relevance to different disease FIGURE 1. CD8+ LCs form a major component of human splenic red states (e.g., ), and to optimize purification for pulp. Top left, middle left, and bottom left panels at increasing original mechanistic investigations, we determined the spectrum of Ags magnification of 320, 3200, and 3400, respectively. CD8 in human splenic expressed (or absent) on human LCs based on study of 20 normal red pulp is highly expressed by the LC population and distributes both at spleens. This was combined with a literature review and a more the cell surface and throughout the cytoplasm (bottom panels). Middle and Downloaded from limited comparison with the LCA. These results are summarized in right panels, No other T cell-specific (CD3, CD4), -specific (CD35, Table II. New observations relevant to the unique phenotype and CD21), /macrophage-associated (CD14, also CD35), or predom- evolution of the human splenic LC, its relationship to the LCA, inant NK cell (CD56) receptors are uniformly expressed by LCs. and the implications of these findings for the regulation of splenic filtration were then further analyzed and are selectively described. vesicle trafficking, plasma membrane blebbing, and downstream signaling to the nucleus (33, 44–46). Most recently, a role in the http://www.jimmunol.org/ LCs express the CD8a/a isoform of CD8 differentiation of smooth muscle cells was uncovered (47, 48). CD8, a coreceptor of the TCR that binds HLA class I, was orig- Reports that FHOD1 could interact with cell surface and transport inally believed to be a unique marker of cytotoxic T cells. Sub- proteins (33, 49) suggested it could also function in relation to sequent investigations revealed certain NK as well as monocyte/ Ag/pathogen or cell invasion/internalization, or in the prevention macrophage and dendritic cells could sometimes express CD8 thereof. Because CD21 was expressed on the LCA (30), when (42). However, the pattern of peptide dimerization (a/b- versus FHOD1 was prominently detected on splenic LCs by in situ hy- a/a-chain) and posttranslational modifications often differed. The bridization (33), we speculated this might relate, in part, to CD21 distribution and composition of CD8 on LCs remained unknown. interaction. However, direct analysis revealed CD21, a known by guest on September 28, 2021 Upon staining of human splenocytes (Fig. 1), prominent CD8 activation Ag, was absent on the normal LC equivalent (Fig. 1). expression on T cells in white pulp as well as on the sinus-lining Using an anti-FHOD1 antiserum produced by our laboratory cells within red pulp was confirmed. There was no significant (Table I), as well as a mouse polyclonal Ab to human FHOD1 staining of red pulp LCs with Ab to CD4 (alternate T/macrophage (Abcam), we did confirm that FHOD1 protein is highly expressed subset), to CD3 (pan T cell), or to CD21 (B cell and follicular by normal LCs but is not expressed by the LCA (Fig. 3). ), despite clear staining of B (CD21) and T cell (CD3, CD4) zones within nearby germinal centers (Fig. 1). CD14+ High expression of DARC distinguishes human LCs from and free CD14 appeared randomly distributed in red adjacent splenocytes, as well as from venous sinus-lining cells pulp and within some sinusoids but did not uniformly associate of other organs with LCs; likewise, CD56, a protein primarily expressed by NK DARC (CD234, gp-Fy) was identified in 1950 as an Ag expressed cells, was not identified on the LC population (Fig. 1). Within red on the surface of human RBCs (50). It was subsequently shown to pulp, cells lining the cords of Billroth and cells within the sinu- be the receptor for and Plasmodium knowlesi, soids were rarely CD8+. In contrast with LCs, these scattered cells as well as a promiscuous receptor for CC and CXC chemokines. showed prominent CD8 membrane expression, whereas LCs ap- DARC is absent on erythrocytes of most West Africans and their peared to accumulate CD8 protein both on the cell membrane descendents because of an RBC-specific promoter polymorphism and diffusely within the cytoplasm (Fig. 1, bottom panels). Using that confers protection from malaria. However, all persons re- a recently available mAb, F-5 (Santa Cruz), that specifically gardless of whether their RBCS are DARC positive or negative, detects human CD8b upon IHC analysis together with a human express DARC on immediate postcapillary endothelial cells and CD8a-specific mAb (Table I), it was apparent that although CD8a certain epithelial cells of , , colon, and brain Purkinje is highly expressed on LCs (Fig. 2C), CD8b (Fig. 2D) is not. cells (50). In spleen, DARC was atypically noted to be highly Control staining of T cells that surround a small is dis- expressed on the sinus-lining cell (34), an observation confirmed played in Fig. 2A (CD8a) and Fig. 2B (CD8b). in this study (Fig. 4); however, no DARC expression could be detected on cells that lined the venous sinusoids of liver and bone High expression of FHOD1 distinguishes LCs from the LCA marrow (34). Similarly, neither CD8 nor FHOD1 was detected In the course of investigation of proteins that interact with the on/in the sinus-lining cells of human liver and (data cytoplasmic domain of CD21, we identified FHOD1 (33), a mem- not shown), emphasizing that a distinct pattern of protein expres- ber of the drosophila-related formin family of actin regulatory sion characterizes the human LC. proteins. Similar to other family members, FHOD1 functions in cytoskeletal regulation through actin cable formation (43). LCs lack CD34, CD45, CD68, and CD163 but express CD31 FHOD1 has further been implicated in dynamic remodeling of CD34 is a marker of most mature cells committed to the endothelial the cytoskeleton, establishment of cell polarity, cell elongation, lineage from which normal LCs are reported to derive. The LCA is The Journal of Immunology 4499

Table I. Abs used for IHC with method information

Detection Ab Clone Species Source Pretreatment System Dilution (1:3) CD3 SP7 RM Thermo Scientific TUF (30 min) RPV 300 CD4 4B12 MM Vector Laboratories EDTA (50 min) MPV 200 CD8 a (human) C8/144B MM Dako EDTA (30 min) MEV 100 CD8 (monkey) VP-C325 Clone 1A5 MM Vector Laboratories Trilogy Solution MEV 50 CD8 b (human) F-5 MM Santa Cruz (sc-25277) EDTA (30 min) MEV 50 CD14 7 MM Abcam (ab49755) EDTA MEV 100 CD19 LE-CD19 MM Serotec (MCA24254T) Citrate MEV 50 CD20 L26 MM Dako (N1502) Dako RTU MEV RTU CD21 IF8 MM Dako (M0784) DAKO PTS (PC 3 min) MPV 40 CD31 JC/70A MM Dako EDTA (30 min) MPV 100 CD34 QBEnd-10 MM Beckman-Coulter Citrate MPV 70 CD35 Ber-MARC-DRC MM; RMa Dako DAKO PTS (PC 3 min) RPV 30 CD45 2B11+PD7/26 MM Dako Citrate MPV 500 CD56 123C3.D5 MM Cell Marque EDTA (30 min) MPV 50 CD68 PGM1 MM Dako (M0876) Dako RTU MEV 200 CD163 10D6 MM NovoCastra DAKO PTS (PC 3 min) MPV 400 CD172a Human (syn. peptide) RP Abcam (ab53721) Citrate REV 2000

CD234 (Darc) 358307 MM R&D Systems (MAB4139) Citrate MEV 150 Downloaded from D2-40 D2-40 MM Covance Citrate (PC) MPV 100 FHOD1 Human protein MP Abcam (ab73443) Citrate MEV 200 (full-length, aa 1–1165) Human (N-terminal aa 1–965) RP Fingeroth Laboratory Thrombospondin 1 A6.1 MM Abcam ab1823 Citrate (PC) MEV 25 (TSP1)

a A mouse anti-human is used as primary followed by a rabbit anti-mouse and then Rabbit Powervision. http://www.jimmunol.org/ MEV, mouse envision; MM, mouse monoclonal, MP, mouse polyclonal; MPV, mouse Powervision; PC, pressure cooker; PTS, pretreatment solution (proprietary); REV, rabbit envision; RM, rabbit monoclonal; RP, rabbit polyclonal; RPV, rabbit Powervision; RTU, ready to use; TUF, tissue unmasking fluid. a low-grade malignancy believed to originate from endothelium, excluded stain. In fact, the pattern of DARC and CD45 expression despite expression of several macrophage-associated Ags. The was nearly reciprocal (Fig. 4A, also see Fig. 5). Thus, the lineage absence of CD34 on LCAs was attributed to changes associated derivation of human splenic LCs (and the LCA) remains inde- with transformation; however, somewhat unexpectedly, CD34 was terminate. LCs did strongly express CD31 (Fig. 4A, top right

also absent on normal LCs (Fig. 4A, top left panel). CD45, in panel), a marker primarily associated with endothelial cells. CD68 by guest on September 28, 2021 contrast, delineates cells that originate from hematopoietic stem (Fig. 4A, bottom middle panel) did not appear to be expressed on cells and is expressed on most mature hematopoietic lineage cells LCs, although it was present on nearby cells (Table II). CD163, with the exception of RBCs. The macrophage-like properties of a common macrophage marker, was detected on cells lining the LCs and expression of CD8 suggested LCs and perhaps the related cords of Billroth (Fig. 4A, bottom right panel), but not on LCs angioma might alternately derive from a hematopoietic precursor. based on evaluation by dual-immunofluorescence analysis with However, although the majority of cells in human spleen bound Abs to both CD163 and CD8 (Fig. 4B). a pan anti-CD45 Ab, the splenic LC population (Fig. 4A, bottom a left panel) along with some small-caliber blood vessels and RBCs LCs express high amounts of SIRP (SHPS-1, BIT, p84, CD172a) SIRPa, a type 1 transmembrane glycoprotein, is primarily ex- pressed on the surface of myeloid lineage (macrophages, dendritic cells, neutrophils) cells (50). It is closely related by structure to the major Ag recognition proteins (50, 51), and like these recep- tors, SIRP genes first appear to coordinate with development of an (52, 53). SIRPa binds its cognate re- ceptor, CD47 (integrin-associated protein), a ubiquitous surface membrane protein and marker of “self,” as well as certain other

FIGURE 2. LCs express the CD8a but not the CD8b-chain of CD8. Immunohistochemical analysis. T cells (control) express both CD8a (A) and CD8b (B), as shown by staining of sequential sections of a periarter- iolar sheath from normal spleen (original magnification 3300). In contrast, the LC network in red pulp stains for CD8a (C), but no CD8b staining (D) FIGURE 3. FHOD1 protein is highly expressed on normal LCs, but not is visualized, although background T cells display dual staining. Also note on the LCA. Immunohistochemical analysis. Expression of FHOD1 on the presence of individual CD8a+ cells between LCs that likely represent normal LCs (left panel) compared with an adjacent section of spleen CD8a/a macrophages (original magnification 3200). containing an LCA (right panel). Original magnification 3200. 4500 HUMAN SPLENIC LITTORAL CELLS Downloaded from FIGURE 5. LCAs retain expression of SIRPa andDARC,andalsolack CD45. (Top panels) Staining of sequential section from an LCA demonstrates that the disorganized sinus-lining cells express both SIRPa (left)andDARC (right). Original magnification 3200. (Middle panels) A reciprocal expression pattern SIRPa+ (left)andCD452 (right) is evident after staining of sequential A FIGURE 4. ( ) LCs do not express either CD34 or CD45, although sections from an LCA. Original magnification 3200. (Bottom left panel)An cellular proteins characteristic of both endothelial and hematopoietic enlarged image demonstrates CD45+ cells between the CD452 sinus-lining http://www.jimmunol.org/ (RBC, macrophage) lineages are variably expressed (CD234, CD31, CD68, cells of an LCA. Original magnification 31000. (Bottom right panel) Dual- CD163). Immunohistochemical analysis. Top left and bottom left panels are immunofluorescent analysis of a segment of normal spleen confirms (as shown lineage markers CD34 and CD45, respectively. Middle top panel is RBC in Fig. 3) that CD45+ cells (red) and LCs (brown) form mutually exclusive marker CD234. Top right panel is endothelial marker CD31. Middle bottom populations, a pattern retained in the LCA. Original magnification 31000. and middle right panels are macrophage markers CD68 and CD163, re- spectively. (B) LCs do not express CD163. Dual-immunofluorescence analysis with anti-CD8 (green) and anti-CD163 (red) reveals that CD163 Is the LCA a bona fide derivative of LCs? does not colocalize with the CD8+ LC, although it is present on nearby cells that line the cords of Billroth. Nuclei are stained blue with DAPI. The LCA is a tumor-like proliferation that occurs only in the by guest on September 28, 2021 Original magnification 3200 (A), 3400 (B). spleen. Frank atypia and metastasis are rare (24, 30, 61). The LCA is believed to derive from normal LCs based on morphologic ap- pearance including disorganized sinuses, debris-filled sinus-lining ligands (37, 54). Interaction with CD47 results in transmission of cells, as well as admixture with a population of taller cells between + a negative intracellular signal that restricts the ability of CD172A and within the sinuses. The appearance of two potentially distinct macrophages to phagocytose and destroy adjacent cells. LPS ex- cell types and an immunophenotype that only partially overlaps posure reduces SIRPa expression, releasing macrophages from (atypically CD82,FHOD12,CD21+,CD68hi) its putative normal quiescence (48) and thereby enhancing their ability to phagocytose counterpart raised questions about whether the LC was the auth- other nearby immune cells to resolve an inflammatory response. entic cell of origin (62). An IHC analysis displayed in Fig. 5 (top Senescent RBCs downregulate CD47, resulting in phagocytosis by panels) showed both SIRPa and DARC were strongly expressed on environmental macrophages. Recently, several human tumor types cells lining the disorganized sinuses of the LCA, and similar to were found to upregulate CD47, providing a relevant mechanism normal LCs, CD45 was absent. These results provide clear evidence forescapefrommacrophageimmunesurveillance(55–57). of the relatedness of the LCA to its normal cell counterpart and Although it has long been recognized that mammalian spleen is establish a firm basis for distinguishing the LCA from other splenic the site where most RBC elimination occurs and furthermore that vascular tumors. turnover is mediated by splenic macrophages, the precise sub- population(s) responsible has remained largely unknown. Recent LC evolution: rodents, nonhuman primates, humans work in mice identified a major requirement for SIRPa and red Much current understanding of splenic function derives from pulp macrophages for efficient RBC phagocytosis (58) and regu- murine studies. However, mice (Mus domesticus) have asinusal lation of iron homeostasis (59). As human and mouse are sepa- spleens in which the red pulp vasculature consists of small, non- rated by 50 million years of evolution, many differences in splenic anastomosing primitive . Rats (Ratus norvegicus) do have structure and immune functions exist; nevertheless, emerging sinusal spleens, as red pulp is filled by large irregular venous evidence suggests major SIRPa functions are conserved (60). To sinuses; however, bona fide endothelial cells with typical lateral identify SIRPa-expressing cells in normal human spleen, we extensions line most of the rat splenic sinuses (15). CD8 expres- performed IHC analyses that revealed that not only were SIRPa- sion in rodent spleen has been well documented; specific Ab binds expressing cells primarily located within the red pulp, but ex- T, as well as NK, monocyte, and macrophage populations in both pression was highly localized to the LC population (Fig. 6, top left rodent’s white and red pulp, but not the venous sinus-lining cell panel). Interestingly, SIRPa expression was also strongly detected (rat). Although the respective genes for murine and rat FHOD1 are on sinus-lining cells in the spleens of Old World primates and predicted to be 85% identical to human FHOD1, we did not detect New World primates, and even rat spleen contained a number of FHOD1 in mouse or rat spleen by RT-PCR (M.B. Gill and J.D. slitlike sinusoids that were lined by cells expressing SIRPa (Fig. 6). Fingeroth, unpublished observations). Furthermore, no FHOD1 The Journal of Immunology 4501

Table II. Multilineage Ag expression pattern of splenic LCs and the LCA

Cell Type Molecule/Marker Functions LC LCA Referencesa Endothelial vWF Factor VIII-related Ag, clotting + + Pusztaszeri et al., 2006 (83) Bi et al., 2007 (62) CD31b PECAM, adhesion, signaling + + Ruck et al., 1994 (84) Arber et al., 1997 (30) Pusztaszeri et al., 2006 (83) CD34b Endothelial lineage marker 22 Pusztaszeri et al., 2006 (83) (also on stem cells) Bi et al., 2007 (62) CD62P P-selectin, adhesion molecule +/22 Korkusuz et al., 2002 (14) CD105 Endoglin, adhesion, and signaling + ND Korkusuz et al., 2002 (14) CD106 VCAM1, adhesion + ND Korkusuz et al., 2002 (14) CD141 Thrombomodulin, thrombin-binding + ND Korkusuz et al., 2002 (14) protein Steininger et al., 2007 (73) CD146 MCAM, adhesion, and cohesion + ND Korkusuz et al., 2002 (14) CD144 VE-cadherin adhesion + ND Pusztaszeri et al., 2006 (83) VEGFR3 FLT4, receptor ND 2 Yamate et al., 2009 (85) D2-40b Lymphatic endothelium marker 22 Korkusuz et al., 2002 (14) Steininger et al., 2004 (72) BMA-120 Unknown glycoprotein + 2 Falk et al., 1991 (86) UEA-1 Ulex europeus Lectin I, binds fucose 2 + Falk et al., 1991 (86) Downloaded from LYVE-1 Lymphatic endothelium hyaluron + ND Martinez-Pomares et al., 2005 (96) receptor Macrophage/monocyte CD172A SIRPa, inhibitory/migratory receptor + + This study HLA II Histocompatibility Ag + + Buckley et al., 1985 (22) Stabilin-1 Scavenger receptor recognizing + ND Goerdt et al., 1991 (36) Toomarian et al., 2011 (87) CD36 Scavenger receptor, phagocytosis + ND Korkusuz et al., 2002 (14) http://www.jimmunol.org/ CD54 ICAM1, adhesion molecule + 2 Korkusuz et al., 2002 (14) CD68b Macrophage marker 2 + Falk et al., 1991 (86) Steininger et al., 2004 (72) Martinez-Pomares et al., 2005 (96) CD163b Macrophage receptor for bacteria 2 + Lau et al., 2004 (88) CD169 Sialoadhesin, adhesion + ND Marmey et al., 2006 (89) CD14 Recognition of LPS, peptidoglycan 2 ND This study CD26 Peptidase + ND Korkusuz et al., 2002 (14) CD35 Complement receptor I 2 ND This study CD11b/CD18 Complement receptor III 2 ND Buckley et al., 1985 (22)

CD71 Transferrin receptor 2 + Buckley et al., 1985 (22) by guest on September 28, 2021 Korkusuz et al., 2002 (14) Ferroportin Iron transporter 2 ND This study CD45 LCA, heme lineage marker 22 This study CD205 DEC, Ag presentation 2 ND Pack et al., 2008 (35) CD206 Macrophage mannose receptor + ND Pusztaszeri et al., 2006 (83) (endocytosis and phagocytosis) CD209 DC-Sign, adhesion receptor 2 ND Bi et al., 2007 (62) Receptor PTPg Phosphatase, suppression inflammation + ND Lissandrini et al., 2006 (90) NK cell CD16 FcgRIIIa and FcgRIIIb 22 Buckley et al., 1991 (99) CD56 NCAM1, adhesion 2 ND This study Dendritic cell Ki-M9 PEST domain protein on follicular dendritic 2 ND Wacker et al., 1997 (95) cells, bone marrow sinus-lining cells RBC CD234b DARC, pan chemokine receptor + + Buckley et al., 1987 (74) T cell CD3b Pan T cell marker 22 Buckley et al., 1985 (22) CD8a HLA I receptor, T and NK cells, + 2 This study macrophages, dendritic cells CD8b T cell coreceptor, HLA I receptor 2 ND This study CD4b T cell coreceptor, helper cell 22 Stuart et al., 1983 (29) CD231 TALLA1, tetraspanin 7 + ND Korkusuz et al., 2002 (14) PD-1 Member extended CD28/CTLA-4 2 ND This study family of T cell regulators CD102 ICAM2, adhesion + ND Korkusuz et al., 2002 (14) B cell CD21b Complement receptor II 2 + Arber et al., 1997 (30) CD19 B cell activation Ag 2 ND This study Bcl-2 Apoptosis regulatory protein 2 ND Jiang et al., 2005 (91) CD20b B cell-restricted surface marker 22 Buckley et al., 1985 (22) Mesenchymal/Smooth FHOD1 Formin, cytoskeletal organization + 2 This study muscle cell SM myosin/myosin Excitation-contraction coupling + ND Pinkus et al., 1986 (97) Drenckhahn et al., 1986 (23) Vimentin Intermediate filament + + Giorno, 1985 (93) Falk et al., 1991 (86) Arber et al., 1997 (30) a-Actinin Stress fiber + ND Drenckhahn et al., 1986 (23) Epithelial cell Cytokeratins, AE1-AE3b Epithelial cytoskeleton 22 Michal et al., 1993 (98) (Table continues) 4502 HUMAN SPLENIC LITTORAL CELLS

Table II. (Continued)

Cell Type Molecule/Marker Functions LC LCA Referencesa Epithelial membrane Agb Pan epithelial marker 22 Michal et al., 1993 (98) Enzymes Lysozyme Muraminidase + + Buckley et al., 1985 (22) a1-Antichymotrypsin Chymotrypsin antagonist + + Falk et al., 1991 (86) Cathepsin D Aspartic proteinase + + Reid et al., 1986 (94) Nonspecific esterase a-Naphthyl acetate and butyrate + + Heusermann and Stutte, 1975 (92) Ruck et al., 1994 (84) Alkaline phosphatase Phosphatase 2 + Heusermann and Stutte, 1975 (92) Megakaryocyte Thrombospondin 1 Cell adhesion, proliferation, motility, 2 ND This study survival Several molecules/markers are multilineage. Table is based on majority literature search. aFor conservation of space, typically only the first literature reference to staining is provided, although several Ab studies were performed and demonstrated in multiple works. bRepresent Ag staining reperformed in this article to clear the discrepancy in the literature. protein was detected in cells forming the sinusoids of rat spleen by Discussion IHC (Fig. 7). Interestingly, rare elongated cells that line a subset of A primordial spleenlike tissue first appeared as a condensation of rat splenic sinusoids did express SIRPa (Fig. 6). These curious the lymphomyeloid complex in the intestine of primitive verte- Downloaded from results suggested that during mammalian evolution, alteration in brates (cyclostomes) (63–65) together with the earliest form of the composition and function of splenic red pulp, specifically RBC, and ever since, their development has remained tightly changes involving the sinus-lining cell, occurred. linked. Although splenic evolution proceeded along a highly In light of the observation that a critical switch from primary variable course relative to location, shape, and function(s), the use of to use of RBC for systemic transport of IC was vascular organization of red pulp (66) remained the major deter-

documented coincident with evolution of Hominidae (9–11), we minant of structure and function. Typical venous sinusoids first http://www.jimmunol.org/ queried whether an unknown, although temporally related, set of arose in mammals (67, 68), but they were only variously adapted events might have driven LC evolution as a cache and/or sieve for by different species; thus, splenic circulation either persisted as the now IC-bearing RBCs. Whereas SIRPa expression was readily typical vascular channels or reorganized as venous lakes (66). In detected on almost all primate sinus-lining cells with the exception the latter case, cells percolate through the red pulp stroma before of Saguinus oedipus (cotton top tamarin) (Fig. 6), FHOD1 was re-entering the circulation on traversing the sinus-lining cell bar- absent on the New World primates Saguinus oedipus and Saimiri rier. Both vessel wall and sinus-lining cells were long assumed to sciureus (squirrel monkey), although moderately expressed on be of endothelial origin. However, with the advent of advanced Callithrix jacchus (common marmoset). All Old World primates light microscopy, Weidenreich (69) and Mollier (70) noted that studied including Macaca mulatta (rhesus), Macaca fascicularis human and certain simian sinus-lining cells were distinct in shape, by guest on September 28, 2021 (cynomolgus), and Chlorocebus sabaeus (African green monkey) were surrounded by annular rings rather than reticulin fiber expressed FHOD1. An mAb, IA5 that detects CD8 on all primates, sheaths, and they lacked the abundant side processes typical of stained scattered cells in the red pulp but did not outline the sinus- mammalian endothelial junctions (reviewed in Refs. 23, 26, 66, lining cell population of any New or Old World primates with 69–71). Application of electron microscopy and development of the exception of chimpanzees (Pan troglodytes), the nonhuman Ag-specific Abs for phenotypic analysis confirmed that these primate closest to human (Fig. 7, top right panel). lining cells were different from those of most other mammals, as well as from the vascular-lining cells (endothelium) of other human organs (29, 72, 73). In 1985, Buckley et al. (22) discovered that the human splenic lining cell or LC atypically expressed CD8, which was then be- lieved to be entirely T cell specific. Buckley et al. confirmed that LCs were CD62 (T cell, scavenger receptor B) and CD202 (B cell), but noted expression of several intracellular enzymes, suggesting a possible macrophage lineage. These results were extended in a subsequent study (74) comparing different macro- phage and dendritic subpopulations in human spleen; and although the phagocytic potential of LCs (nonspecific esterase positive, lysozyme positive) and other monocyte/macrophage-like charac- teristics (HLA-DR+, CD36+) were described (see Table II), LCs remained classified as endothelial cells (23, 45). Despite development of many new reagents for delineation of cellular phenotype, few studies in the intervening decades focused on the human LC. This was notwithstanding a dominant presence FIGURE 6. SIRPa is highly expressed on human splenic LCs and is in red pulp and .100 years of accumulated microscopy sug- variably detected on primate and rodent sinus-lining cells. Human LCs gesting LCs formed a uniquely selective barrier that filtered (top left panel) as well as the sinus-lining cells of MM (Old World mon- key) (top right panel) express high amounts of SIRPa, whereas expression RBCs (senescent, pathogen infected), other leukocytes, and altered among SgO (New World monkey) (bottom left panel) is more subtle. Most self. Consequently, mechanistic information is lacking, and in sinus-lining cells in rat spleen do not express SIRPa; however, a subpop- vitro culture systems do not exist. The LCA, a rare proliferative ulation that does express SIRPa can be detected (bottom right panel). lesion found only in human spleen, is characterized by disorga- Original magnification 3200. nized sinus-lining cells, few atypical mitoses, and is curiously The Journal of Immunology 4503

FIGURE 7. The CD8+ splenic LC is an evolution- ary newcomer. Top left panels, FHOD1 is expressed in sinus-lining cells of humans and MM (Old World monkey), but cannot be detected in SgO (New World monkey) or in rats. Top right panels, CD8 is highly expressed on the sinus-lining cells of a chimpanzee (Hominidae), whereas no CD8 protein is detected on the sinus-lining cells of a baboon (Old World mon- key). Bottom, Summary and diagram of expression patterns that describe LC evolution. Original magni- fication 3200. Downloaded from http://www.jimmunol.org/ associated with unrelated tumors at distant sites. Although the ramigration) as well as through (transmigration) LCs or are de- LCA was believed to originate from LCs, many of the applied cell stroyed within the LC is consistent with a major role in regulating markers were discordant, posing diagnostic difficulty when com- the cellular composition of the systemic circulation. We speculate pared with other splenic vascular tumors (S.J. Rodig, manuscript that similar to a customs checkpoint, the panel of Ags expressed on in preparation). Therefore, we reviewed the published literature LCs initially determine which cells (documents) are appropriate for and analyzed, as well as reanalyzed, the expression pattern of passage, then recheck their content (baggage) and finally engulf the normal human LC and the LCA (Table II), providing salient (reject) or contract (open the gate), permitting entry into the cir- new information for distinguishing and isolating LCs from other culation (country). Multiple cell–cell interactions are surely in- splenic populations. Our findings verify the relatedness of the volved that we have just begun to identify. The significance of by guest on September 28, 2021 LCA and LC, because SIRPa and DARC are highly expressed CD8 expression is uncertain, although it is notable that RBCs do on both normal and angiomatous sinus-lining cells, whereas the not express and many tumors downmodulate HLA I, suggesting markers CD8 (2), FHOD1 (2), and CD21 (+) discriminate neo- that the absence of a “self” interaction signals a requirement for plastic from normal cells. additional review. Evidence already exists that, similar to mice, The high expression and the unique distribution of CD8a/a, the SIRPa-CD47 axis in human is of major import in regulating FHOD1, and SIRPa, together with DARC and specific subsets of the turnover of RBCs, as well as survival of stem cells and tumor adhesion and phagocytic/endocytic receptors (e.g., stabilin-1) re- cells (55, 80, 81). Upon arrival at the LC periphery, failure to cently implicated in phosphatidylserine-dependent phagocytosis engage SIRPa on the basis of reduced CD47 (e.g., typical of of RBCs and cell corpses in mice (36, 75, 76) provide much in the senescent RBCs) would stimulate an “eat me” signal leading to cell way of new insights into the likely mechanisms underlying the destruction, whereas the absence of such a signal could facilitate function(s) of the human LC, a cell that appears in current form passage to the sinus. In recent work, the actin nucleator FHOD1 was (CD8a/a+) only upon evolution of Hominidae (humans, chimps). discovered to be activated downstream of RhoA-ROCK (in addition Because splenic and RBC evolution are historically tightly linked to Rac), thereby promoting development of a smooth muscle cell (63, 66), it is particularly noteworthy that coincident with the phenotype with contractile function (23, 47). Thus, upon delivery rapid evolution of the LC, systemic transport of ICs switches from of an integrated checkpoint signal from ligation of multireceptor platelets to RBCs accompanied by acquisition of RBC comple- surface complexes, contraction of LCs might promote rapid local ment receptor type 1/CD35 (11). The LC appears well equipped to release of cells stored in spleen, such as RBCs, tumor cells, or process membrane-altered RBCs or RBCs that bear residual ICs perhaps global release of monocytes for cardiac repair, as recently (particularly if ICs were not eliminated during earlier stages of described in murine spleen (82). The multilineage characteristics or hepatic or splenic transport), because its most important role plasticity of LCs as demonstrated by expression of a spectrum of may be sorting of the cleared RBC. Many of the described LC Ags associated with endothelial, monocyte/macrophage/dendritic, surface receptors (Table II) have known adhesive and/or phago- as well as smooth muscle cells reveals a newly arrived and highly cytic functions; however, it is notable that the subset of receptors specialized barrier cell whose role as a major component of human currently identified on LCs predominantly predicts anti-inflamma- spleen awaits precise definition. tory rather than proinflammatory response patterns (e.g., DARC, CD206, CD163, CD31, enzymes, chemokines) (37, 77, 78, re- Acknowledgments viewed in Ref. 79). We sincerely thank the donor families and the staff of the New England The strategic location of the LC compartment and electron Organ Bank, Scott Johnson, Seth Karp, Amy Evenson, and Douglas Hanto microscopic evidence that RBCs and other cells (lymphocytes, of the Department of Surgery (Beth Israel Deaconess Medical Center) for other mononuclear cells, possibly tumor cells) pass between (pa- support. We thank Christine Unitt, Corey Marvin, Tyler Caron, Mei Zheng, 4504 HUMAN SPLENIC LITTORAL CELLS

Jason Hornick, Qian Zhan, Alyson Smeedy Campbell, Sara Akhavandord, 30. Arber, D. A., J. G. Strickler, Y. Y. Chen, and L. M. Weiss. 1997. Splenic vascular and John Iafrate for advice and/or technical assistance. We thank Dr. tumors: a histologic, immunophenotypic, and virologic study. Am. J. Surg. Pathol. 21: 827–835. Michael Gimbrone (Harvard Medical School), Prof. Dyann Wirth (Harvard 31. Bisceglia, M., J. Z. Sickel, F. Giangaspero, V. Gomes, M. Amini, and M. Michal. School of Public Health), and Prof. Terrie Taylor (Michigan State Uni- 1998. Littoral cell angioma of the spleen: an additional report of four cases with versity) for support and mentorship of D.A.M. for this work. We thank emphasis on the association with visceral organ cancers. Tumori 84: 595–599. Dr. Anne Nicholson-Weller for valuable comments and Dr. Johanna Daily 32. Gupta, M. K., M. Levin, N. S. Aguilera, and G. M. Pastores. 2001. Littoral cell angioma of the spleen in a patient with Gaucher disease. Am.J.Hematol.68: 61–62. for continued collaboration. 33. Gill, M. B., J. Roecklein-Canfield, D. R. Sage, M. Zambela-Soediono, N. Longtine, M. Uknis, and J. D. Fingeroth. 2004. EBV attachment stimulates FHOS/FHOD1 redistribution and co-aggregation with CD21: formin interactions with the cyto- Disclosures plasmic domain of human CD21. J. Cell Sci. 117: 2709–2720. The authors have no financial conflicts of interest. 34. Peiper, S. C., Z. X. Wang, K. Neote, A. W. Martin, H. J. Showell, M. J. Conklyn, K. Ogborne, T. J. Hadley, Z. H. Lu, J. Hesselgesser, and R. Horuk. 1995. The Duffy /receptor for chemokines (DARC) is expressed in endothelial cells References of Duffy negative individuals who lack the erythrocyte receptor. J. Exp. Med. 1. Patel, S., N. Kramer, and E. D. Rosenstein. 2010. Evolving connective tissue 181: 1311–1317. disease influenced by : beneath the sword of Dameshek. J. Clin. 35. Pack, M., C. Trumpfheller, D. Thomas, C. G. Park, A. Granelli-Piperno, Rheumatol. 16: 280–283. C. Mu¨nz, and R. M. Steinman. 2008. DEC-205/CD205+ dendritic cells are 2. Linet, M. S., O. Nyre´n, G. Gridley, H. O. Adami, J. D. Buckland, J. K. McLaughlin, abundant in the white pulp of the human spleen, including the border region and J. F. Fraumeni, Jr. 1996. Causes of death among patients surviving at least between the red and white pulp. Immunology 123: 438–446. one year following splenectomy. Am. J. Surg. 172: 320–323. 36. Goerdt, S., L. J. Walsh, G. F. Murphy, and J. S. Pober. 1991. Identification of 3. Robinette, C. D., and J. F. Fraumeni, Jr. 1977. Splenectomy and subsequent a novel high molecular weight protein preferentially expressed by sinusoidal mortality in veterans of the 1939-45 war. Lancet 2: 127–129. endothelial cells in normal human tissues. J. Cell Biol. 113: 1425–1437. 4. Cesta, M. F. 2006. Normal structure, function, and histology of the spleen. 37. Taylor, P. R., L. Martinez-Pomares, M. Stacey, H. H. Lin, G. D. Brown, and Toxicol. Pathol. 34: 455–465. S. Gordon. 2005. Macrophage receptors and immune recognition. Annu. Rev. Downloaded from 5. Rosse, W. F. 1987. The spleen as a filter. N. Engl. J. Med. 317: 704–706. Immunol. 23: 901–944. 6. Brozman, M., and J. Jakubovsky´. 1989. The red pulp of the human spleen. 38. van den Berg, T. K., and C. E. van der Schoot. 2008. Innate immune ‘self’ Structural basis of blood filtration. Z. Mikrosk. Anat. Forsch. 103: 316–328. recognition: a role for CD47-SIRPalpha interactions in 7. Hart, S. P., J. R. Smith, and I. Dransfield. 2004. Phagocytosis of opsonized ap- transplantation. Trends Immunol. 29: 203–206. optotic cells: roles for ‘old-fashioned’ receptors for and complement. 39. Kinchen, J. M., and K. S. Ravichandran. 2008. Phagocytic signaling: you can Clin. Exp. Immunol. 135: 181–185. touch, but you can’t eat. Curr. Biol. 18: R521–R524. 8. Fearon, D. T. 1980. Identification of the membrane glycoprotein that is the C3b 40. Rodig, S. J., J. Ouyang, P. Juszczynski, T. Currie, K. Law, D. S. Neuberg,

receptor of the human erythrocyte, polymorphonuclear leukocyte, B , G. A. Rabinovich, M. A. Shipp, and J. L. Kutok. 2008. AP1-dependent galectin- http://www.jimmunol.org/ and monocyte. J. Exp. Med. 152: 20–30. 1 expression delineates classical hodgkin and anaplastic large cell lymphomas 9. Hebert, L. A. 1991. The clearance of immune complexes from the circulation of from other lymphoid malignancies with shared molecular features. Clin. Cancer man and other primates. Am. J. Kidney Dis. 17: 352–361. Res. 14: 3338–3344. 10. Nelson RA, Jr. 1953. The immune-adherence phenomenon; an immunologically 41. Yearley, J. H., C. Pearson, R. P. Shannon, and K. G. Mansfield. 2007. Phenotypic specific reaction between microorganisms and erythrocytes leading to enhanced variation in myocardial macrophage populations suggests a role for macrophage phagocytosis. Science 118: 733–737. activation in SIV-associated cardiac disease. AIDS Res. Hum. Retroviruses 23: 11. Birmingham, D. J., and L. A. Hebert. 2001. CR1 and CR1-like: the primate 515–524. immune adherence receptors. Immunol. Rev. 180: 100–111. 42. Gibbings, D. J., M. Marcet-Palacios, Y. Sekar, M. C. Ng, and A. D. Befus. 2007. 12. Jacobson, A. C., and J. H. Weis. 2008. Comparative functional evolution of CD8 alpha is expressed by human monocytes and enhances Fc gamma R- human and mouse CR1 and CR2. J. Immunol. 181: 2953–2959. dependent responses. BMC Immunol. 8: 12. 13. Hirasawa, Y., and H. Tokuhiro. 1970. Electron microscopic studies on the normal 43. Faix, J., and R. Grosse. 2006. Staying in shape with formins. Dev. Cell 10: 693–

human spleen: especially on the red pulp and the reticulo-endothelial cells. 706. by guest on September 28, 2021 Blood 35: 201–212. 44. Gasteier, J. E., S. Schroeder, W. Muranyi, R. Madrid, S. Benichou, and 14. Korkusuz, P., A. Dağdeviren, and E. Aşan. 2002. Immunophenotypic analysis of O. T. Fackler. 2005. FHOD1 coordinates actin filament and microtubule align- human spleen compartments. Ann. Anat. 184: 431–441. ment to mediate cell elongation. Exp. Cell Res. 306: 192–202. 15. Udroiu, I. 2006. Evolution of sinusal and non-sinusal spleens of mammals. 45. Hannemann, S., R. Madrid, J. Stastna, T. Kitzing, J. Gasteier, A. Scho¨nichen, Hystrix. 17: 99–116. J. Bouchet, A. Jimenez, M. Geyer, R. Grosse, et al. 2008. The Diaphanous- 16. Hataba, Y., Y. Kirino, and T. Suzuki. 1981. Scanning electron microscopic study related Formin FHOD1 associates with ROCK1 and promotes Src-dependent of the red pulp of mouse spleen. J. Electron Microsc. (Tokyo) 30: 46–56. plasma membrane blebbing. J. Biol. Chem. 283: 27891–27903. 17. Snook, T. 1950. A comparative study of the vascular arrangements in mam- 46. Young, K. G., and J. W. Copeland. 2010. Formins in cell signaling. Biochim. malian spleens. Am. J. Anat. 87: 31–77. Biophys. Acta 1803: 183–190. 18. Weiss, L. 1983. The red pulp of the spleen: structural basis of blood flow. Clin. 47. Staus, D. P., A. L. Blaker, M. D. Medlin, J. M. Taylor, and C. P. Mack. 2011. Haematol. 12: 375–393. Formin homology domain-containing protein 1 regulates smooth muscle cell 19. Schmidt, E. E., I. C. MacDonald, and A. C. Groom. 1985. Microcirculation in rat phenotype. Arterioscler. Thromb. Vasc. Biol. 31: 360–367. (sinusal) spleen studied by means of corrosion casts, with particular reference to 48. Kong, X. N., H. X. Yan, L. Chen, L. W. Dong, W. Yang, Q. Liu, L. X. Yu, intermediate pathways. J. Morphol. 186: 1–16. D. D. Huang, S. Q. Liu, H. Liu, et al. 2007. LPS-induced down-regulation of 20. Schmidt, E. E., I. C. MacDonald, and A. C. Groom. 1985. Microcirculation in signal regulatory protein alpha contributes to innate immune activation in mouse (nonsinusal) spleen studied by means of corrosion casts. J. Morphol. 186: macrophages. J. Exp. Med. 204: 2719–2731. 17–29. 49. Tojo, H., I. Kaieda, H. Hattori, N. Katayama, K. Yoshimura, S. Kakimoto, Y., 21. McCuskey, R. S., and P. A. McCuskey. 1985. In vivo and electron microscopic E. Presman, C. C. Brooks, and P. F. Pilch. 2003. The Formin family protein, studies of the splenic microvasculature in mice. Experientia 41: 179–187. formin homolog overexpressed in spleen, interacts with the insulin-responsive 22. Buckley, P. J., S. A. Dickson, and W. S. Walker. 1985. Human splenic sinusoidal aminopeptidase and profilin IIa. Mol. Endocrinol. 17: 1216–1229. lining cells express associated with monocytes, macrophages, endo- 50. Barclay, A. N. 2009. Signal regulatory protein alpha (SIRPalpha)/CD47 inter- thelial cells, and T lymphocytes. J. Immunol. 134: 2310–2315. action and function. Curr. Opin. Immunol. 21: 47–52. 23. Drenckhahn, D., and J. Wagner. 1986. Stress fibers in the splenic sinus endo- 51. Hatherley, D., S. C. Graham, K. Harlos, D. I. Stuart, and A. N. Barclay. 2009. thelium in situ: molecular structure, relationship to the extracellular matrix, and Structure of signal-regulatory protein alpha: a link to antigen receptor evolution. contractility. J. Cell Biol. 102: 1738–1747. J. Biol. Chem. 284: 26613–26619. 24. Kutok, J. L., and C. D. Fletcher. 2003. Splenic vascular tumors. Semin. Diagn. 52. van den Berg, T. K., J. A. Yoder, and G. W. Litman. 2004. On the origins of Pathol. 20: 128–139. adaptive immunity: innate immune receptors join the tale. Trends Immunol. 25: 25. Barnhart, M. I., and J. M. Lusher. 1976. The human spleen as revealed by 11–16. scanning electron microscopy. Am. J. Hematol. 1: 243–264. 53. van Beek, E. M., F. Cochrane, A. N. Barclay, and T. K. van den Berg. 2005. 26. Fujita, T., M. Kashimura, and K. Adachi. 1982. Scanning electron microscopy Signal regulatory proteins in the immune system. J. Immunol. 175: 7781–7787. (SEM) studies of the spleen—normal and pathological. Scan. Electron Microsc. 54. Sarfati, M., G. Fortin, M. Raymond, and S. Susin. 2008. CD47 in the immune (Pt 1): 435–444. response: role of thrombospondin and SIRP-alpha reverse signaling. Curr. Drug 27. Pongponratn, E., M. Riganti, D. Bunnag, and T. Harinasuta. 1987. Spleen in Targets 9: 842–850. falciparum malaria: ultrastructural study. Southeast Asian J. Trop. Med. Public 55. Jaiswal, S., M. P. Chao, R. Majeti, and I. L. Weissman. 2010. Macrophages as Health 18: 491–501. mediators of tumor immunosurveillance. Trends Immunol. 31: 212–219. 28. Jiskoot, P. M., C. Halsey, R. Rivers, B. J. Bain, and B. S. Wilkins. 2004. Unusual 56. Majeti, R., M. P. Chao, A. A. Alizadeh, W. W. Pang, S. Jaiswal, K. D. Gibbs, Jr., splenic sinusoidal iron overload in sickle cell/haemoglobin D-Punjab disease. N. van Rooijen, and I. L. Weissman. 2009. CD47 is an adverse prognostic factor J. Clin. Pathol. 57: 539–540. and therapeutic antibody target on human acute myeloid stem cells. 29. Stuart, A. E., and A. Warford. 1983. Staining of human splenic sinusoids and Cell 138: 286–299. demonstration of unusual banded structures by monoclonal antisera. J. Clin. 57. Chan, K. S., I. Espinosa, M. Chao, D. Wong, L. Ailles, M. Diehn, H. Gill, Pathol. 36: 1176–1180. J. Presti, Jr., H. Y. Chang, M. van de Rijn, et al. 2009. Identification, molecular The Journal of Immunology 4505

characterization, clinical prognosis, and therapeutic targeting of human bladder 81. Takenaka, K., T. K. Prasolava, J. C. Wang, S. M. Mortin-Toth, S. Khalouei, tumor-initiating cells. Proc. Natl. Acad. Sci. USA 106: 14016–14021. O. I. Gan, J. E. Dick, and J. S. Danska. 2007. Polymorphism in Sirpa modulates 58. Oldenborg, P. A., A. Zheleznyak, Y. F. Fang, C. F. Lagenaur, H. D. Gresham, and engraftment of human hematopoietic stem cells. Nat. Immunol. 8: 1313–1323. F. P. Lindberg. 2000. Role of CD47 as a marker of self on red blood cells. 82. Swirski, F. K., M. Nahrendorf, M. Etzrodt, M. Wildgruber, V. Cortez-Retamozo, Science 288: 2051–2054. P. Panizzi, J. L. Figueiredo, R. H. Kohler, A. Chudnovskiy, P. Waterman, et al. 59. Kohyama, M., W. Ise, B. T. Edelson, P. R. Wilker, K. Hildner, C. Mejia, 2009. Identification of splenic reservoir monocytes and their deployment to in- W. A. Frazier, T. L. Murphy, and K. M. Murphy. 2009. Role for Spi-C in the flammatory sites. Science 325: 612–616. development of red pulp macrophages and splenic iron homeostasis. Nature 457: 83. Pusztaszeri, M. P., W. Seelentag, and F. T. Bosman. 2006. Immunohistochemical 318–321. expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 60. Tsai, R. K., P. L. Rodriguez, and D. E. Discher. 2010. Self inhibition of phago- in normal human tissues. J. Histochem. Cytochem. 54: 385–395. cytosis: the affinity of ‘marker of self’ CD47 for SIRPalpha dictates potency of 84. Ruck, P., E. Kaiserling, H. P. Horny, K. Marzusch, J. A. Dietl, A. Geiselhart, inhibition but only at low expression levels. Blood Cells Mol. Dis. 45: 67–74. R. Handgretinger, G. Vince, and C. W. Redman. 1994. Cell adhesion molecules 61. Fernandez, S., G. W. Cook, and D. A. Arber. 2006. Metastasizing splenic littoral on large granular lymphocytes and endothelial cells in decidua of early human cell . Am. J. Surg. Pathol. 30: 1036–1040. . Virchows Arch. 424: 228. 62. Bi, C. F., L. L. Jiang, Z. Li, and W. P. Liu. 2007. [Littoral cell angioma of spleen: 85. Yamate, J., T. Izawa, M. Kuwamura, F. Mitsunaga, and S. Nakamura. 2009. a clinicopathologic study of 17 cases]. Zhonghua Bing Li Xue Za Zhi 36: 239–243. Vasoformative disorder, resembling littoral cell angioma, of the spleen in a ge- 63. Glomski, C. A., J. Tamburlin, and M. Chainani. 1992. The phylogenetic odyssey riatric Japanese macaque (Macaca fuscata). Vet. Pathol. 46: 520–525. of the erythrocyte. III. , the lower experience. Histol. Histopathol. 86. Falk, S., H. J. Stutte, and G. Frizzera. 1991. Littoral cell angioma. A novel 7: 501–528. splenic vascular lesion demonstrating histiocytic differentiation. Am. J. Surg. 64. Tanaka, Y., Y. Saito, and H. Gotoh. 1981. Vascular architecture and intestinal he- Pathol. 15: 1023–1033. matopoietic nests of two cyclostomes, Eptatretus burgeri and ammoncoetes of 87. Toomarian, L., M. Sattari, N. Hashemi, N. Tadayon, and A. Akbarzadeh Entosphenus reissneri: a comparative morphological study. J. Morphol. 170: 71–93. Baghban. 2011. Comparison of neutrophil apoptosis, alpha-defensins and cal- 65. Jønsson, V. 1985. Comparison and definition of spleen and : a phy- protectin in children with and without severe early childhood caries. Iran J. logenetic analysis. J. Theor. Biol. 117: 691–699. Immunol. 8: 11–19. 66. Tischendorf, F. 1985. On the evolution of the spleen. Experientia 41: 145–152. 88. Lau, S. K., P. G. Chu, and L. M. Weiss. 2004. CD163: a specific marker of 67. Hartman, A. 1930. Die Milz. In Handbuch der Mikroskopischen Anatomie des macrophages in paraffin-embedded tissue samples. Am. J. Clin. Pathol. 122: Downloaded from Menschen 6 ( Pt 1). Springer, Berlin, p. 397–563. 794–801. 68. Murata, H. 1959. Comparative studies of the spleen in submammalian verte- 89. Marmey, B., C. Boix, J. B. Barbaroux, M. C. Dieu-Nosjean, J. Diebold, brates. Part II. Minute structure of the spleen, with special reference to the J. Audouin, W. H. Fridman, C. G. Mueller, and T. J. Molina. 2006. CD14 and periarterial lymphoid sheath. Bull. Yamaguchi Med. Sch. 6: 83–105. CD169 expression in human lymph nodes and spleen: specific expansion of 69. Weidenreich, E. 1901. Das Gefaßsystem der menschlischen Milz. Arch. f. mikr. CD141CD169- monocyte-derived cells in diffuse large B-cell lymphomas. Anat. u Entwick. 58: 247–376. Hum. Pathol. 37: 68–77. 70. Mollier, S. 1911. Uber den Bau der capillaren Milzvenen (Milzsinus). Arch. 90. Lissandrini, D., W. Vermi, M. Vezzalini, S. Sozzani, F. Facchetti, G. Bellone,

f. mikr. Anat. 76: 608–657. A. Mafficini, F. Gentili, M. G. Ennas, C. Tecchio, and C. Sorio. 2006. Receptor- http://www.jimmunol.org/ 71. Fujita, T., M. Kashimura, and K. Adachi. 1985. Scanning electron microscopy type protein tyrosine phosphatase gamma (PTPgamma), a new identifier for and terminal circulation. Experientia 41: 167–179. myeloid dendritic cells and specialized macrophages. Blood 108: 4223–4231. 72. Steininger, H., D. Pfofe, L. Marquardt, H. Sauer, and R. Markwat. 2004. Isolated 91. Jiang, L., D. Jones, L. J. Medeiros, Y. R. Orduz, and C. E. Bueso-Ramos. 2005. diffuse hemangiomatosis of the spleen: case report and review of literature. Peripheral T-cell lymphoma with a “follicular” pattern and the perifollicular Pathol. Res. Pract. 200: 479–485. sinus phenotype. Am. J. Clin. Pathol. 123: 448–455. 73. Steininger, B., V. Stachniss, H. Schwarzbach, and P. J. Barth. 2007. Phenotypic 92. Heusermann, U., and H. J. Stutte. 1975. Comparative histochemical and electron differences between red pulp and sinusoidal endothelia help localizing microscopic studies of the sinus and venous walls of the human spleen with the open splenic circulation in humans. Histochem. Cell Biol. 128: 391–398. special reference to the sinus-venous connections. Cell Tissue Res. 163: 519– 74. Buckley, P. J., M. R. Smith, M. F. Braverman, and S. A. Dickson. 1987. Human 533. spleen contains phenotypic subsets of macrophages and dendritic cells that oc- 93. Giorno, R. 1985. Immunohistochemical analysis of the distribution of vimentin cupy discrete microanatomic locations. Am. J. Pathol. 128: 505–520. in human peripheral lymphoid tissues. Anat. Rec. 211: 43–47.

75. Lee, S. J., Park, S. Y., Jung, M. Y., Bae, S. M., and Kim, I. S. 2011. Mechanism 94. Reid, W. A., M. J. Valler, and J. Kay. 1986. Immunolocalization of cathepsin D by guest on September 28, 2021 for phosphatidylserine-dependent erythrophagocytosis in mouse liver. Blood. in normal and neoplastic human tissues. J. Clin. Pathol. 39: 1323–1330. 117: 5215–5223. 95. Wacker, H. H., S. O. Frahm, H. J. Heidebrecht, and R. Parwaresch. 1997. Sinus- 76. Park, S. Y., M. Y. Jung, S. J. Lee, K. B. Kang, A. Gratchev, V. Riabov, lining cells of the lymph nodes recognized as a dendritic cell type by the new J. Kzhyshkowska, and I. S. Kim. 2009. Stabilin-1 mediates phosphatidylserine- monoclonal antibody Ki-M9. Am. J. Pathol. 151: 423–434. dependent clearance of cell corpses in alternatively activated macrophages. 96. Martinez-Pomares, L., L. G. Hanitsch, R. Stillion, S. Keshav, and S. Gordon. J. Cell Sci. 122: 3365–3373. 2005. Expression of mannose receptor and ligands for its cysteine-rich domain in 77. Afenyi-Annan, A., M. Kail, M. R. Combs, E. P. Orringer, A. Ashley-Koch, and venous sinuses of human spleen. Lab. Invest. 85: 1238–1249. M. J. Telen. 2008. Lack of Duffy antigen expression is associated with organ 97. Pinkus, G. S., M. J. Warhol, E. M. O’Connor, C. L. Etheridge, and K. Fujiwara. damage in patients with sickle cell disease. Transfusion 48: 917–924. 1986. Immunohistochemical localization of smooth muscle myosin in human 78. Privratsky, J. R., D. K. Newman, and P. J. Newman. 2010. PECAM-1: conflicts spleen, lymph node, and other lymphoid tissues. Unique staining patterns in of interest in inflammation. Life Sci. 87: 69–82. splenic white pulp and sinuses, lymphoid follicles, and certain vasculature, with 79. Chakera, A., R. M. Seeber, A. E. John, K. A. Eidne, and D. R. Greaves. 2008. ultrastructural correlations. Am. J. Pathol. 123: 440–453. The duffy antigen/receptor for chemokines exists in an oligomeric form in living 98. Michal, M., A. Skalova, F. Fakan, V. Koza, and M. Svojgrova. 1993. Littoral cell cells and functionally antagonizes CCR5 signaling through hetero-oligomeri- angioma of the spleen. A case report with ultrastructural and immunohisto- zation. Mol. Pharmacol. 73: 1362–1370. chemical observations. Zentralbl. Pathol. 139: 361–365. 80. Garratty, G. 2008. The James Blundell Award Lecture 2007: do we really un- 99. Buckley, P. J. 1991. Phenotypic subpopulations of macrophages and dendritic derstand immune red cell destruction? Transfus. Med. 18: 321–334. cells in human spleen. Scanning Microsc. 5: 147–157; discussion 157–148.