Structures of LRP2 reveal a molecular machine for endocytosis.

The low-density lipoprotein (LDL) receptor-related protein 2 (LRP2 or megalin) is representative of the phylogenetically conserved subfamily of giant LDL receptor-related proteins, which function in endocytosis and are implicated in diseases of the kidney and brain. Here, we report high-resolution cryoelectron microscopy structures of LRP2 isolated from mouse kidney, at extracellular and endosomal pH. The structures reveal LRP2 to be a molecular machine that adopts a conformation for ligand binding at the cell surface and for ligand shedding in the endosome. LRP2 forms a homodimer, the conformational transformation of which is governed by pH-sensitive sites at both homodimer and intra-protomer interfaces. A subset of LRP2 deleterious missense variants in humans appears to impair homodimer assembly. These observations lay the foundation for further understanding the function and mechanism of LDL receptors and implicate homodimerization as a conserved feature of the LRP receptor subfamily.

Agents of cancer immunosurveillance: HSPs and dsDNA.

Tumor immunosurveillance requires tumor cell-derived molecules to initiate responses through corresponding receptors on antigen presenting cells (APCs) and a specific effector response designed to eliminate the emerging tumor cells. This is supported by evidence from immunodeficient individuals and experimental animals. Recent discoveries suggest that adjuvanticity of tumor-derived heat shock proteins (HSPs) and double-stranded DNA (dsDNA) are necessary for tumor-specific immunity. There is also the obligatory early transfer of tumor antigens to APCs. We argue that tumor-derived HSPs deliver sufficient chaperoned antigen for cross-priming within the quantitative limits set by nascent tumors. In contrast to late-stage tumors, we are only just beginning to understand the unique interactions of the immune system with precancerous/nascent neoplastic cells, which is important for improved cancer prevention measures.

A reactive monocyte subset characterized by low expression of CD91 is expanded during sterile and septic inflammation.

OBJECTIVES: This study was undertaken to assess CD91 expression on monocytes and changes in monocyte subset distribution during acute tissue damage and bloodstream infection (BSI). METHODS: We investigated blood specimens from healthy individuals, trauma and cardiac surgery patients as a model of tissue damage, and patients with BSI, by flow cytometry using a panel of antibodies comprising CD45, HLA-DR, CD14, CD16 and CD91 for the identification of monocyte subsets. RESULTS: While infrequent in healthy subjects, CD91low/neg monocyte levels were markedly high in BSI, trauma and after cardiac surgery. This monocyte subset expanded up to 15-fold in both patient cohorts, whereas CD14+CD16+ inflammatory monocytes were multiplied by a factor of 5 only. CD14+CD91low monocytes displayed a significantly lower density of HLA-DR and markedly reduced expression of CD300e, compared to the other subsets. They also expressed high levels of myeloperoxidase and showed robust phagocytic and oxidative burst activity. CONCLUSIONS: Expansion of CD91low monocytes is a sensitive marker of acute inflammatory states of infectious and non-infectious etiology.

Leukocyte surface biomarkers implicate deficits of innate immunity in sporadic Alzheimer's disease.

INTRODUCTION: Blood-based diagnostics and prognostics in sporadic Alzheimer's disease (AD) are important for identifying at-risk individuals for therapeutic interventions. METHODS: In three stages, a total of 34 leukocyte antigens were examined by flow cytometry immunophenotyping. Data were analyzed by logistic regression and receiver operating characteristic (ROC) analyses. RESULTS: We identified leukocyte markers differentially expressed in the patients with AD. Pathway analysis revealed a complex network involving upregulation of complement inhibition and downregulation of cargo receptor activity and Aß clearance. A proposed panel including four leukocyte markers - CD11c, CD59, CD91, and CD163 - predicts patients' PET Aß status with an area under the curve (AUC) of 0.93 (0.88 to 0.97). CD163 was the top performer in preclinical models. These findings have been validated in two independent cohorts. CONCLUSION: Our finding of changes on peripheral leukocyte surface antigens in AD implicates the deficit in innate immunity. Leukocyte-based biomarkers prove to be both sensitive and practical for AD screening and diagnosis.

LRP1/CD91 is highly expressed in monocytes from patients with vitiligo - even after repigmentation.

Vitiligo pathophysiology is mediated by antigen-specific cytotoxic T cells. Environmental stressors cause susceptible melanocytes to secrete damage-associated molecular patterns (DAMPs). DAMPs are recognized by receptors such as the endocytic low-density lipoprotein receptor-related protein (LRP1/CD91), expressed in antigen-presenting cells, which activate self-reactive CD8+ T cells, leading to melanocyte destruction. Within this response, interferon gamma triggers production of cytokine CXCL10, recruiting more activated T cells causing further melanocytic damage. We hypothesized that expression of LRP1/CD91 was higher in vitiligo patients compared to non-vitiligo individuals. And further that levels/expression of CXCL10 in plasma were linked to disease severity. We enrolled forty individuals in this study: 18 patients with vitiligo and 22 healthy volunteers. We assessed LRP1/CD91 expression and plasma CXCL10 in patients with vitiligo and healthy volunteers. Additionally, vitiligo patients received combined treatment for 16 weeks following which the said parameters were reassessed. Vitiligo Area Scoring Index was calculated before and after treatment for these patients. Analysis of LRP1/CD91 MFI values in monocytes from vitiligo patients showed high surface levels of LRP1/CD91 than from healthy volunteers (10.50 ± 0.77 vs. 6.55 ± 0.77 MFI units, p < 0.001). This expression did not change after treatment. Plasma levels of CXCL10 were higher in vitiligo patients than healthy volunteers (93.78 ± 7.73 vs. 40.17 ± 6.25 pg/ml). The patients with a good clinical response to treatment had a parallel reduction in plasma CXCL10 levels (105.8 ± 18.44 vs. 66.13 ± 4.87 pg/ml) before and after treatment. LRP1/CD91 expression may reflect susceptibility to vitiligo. Plasma levels of CXCL10 can represent a biomarker for monitoring treatment response. LRP1 and CXCL10 may represent therapeutic targets.

CD91 Derived Treg Epitope Modulates Regulatory T Lymphocyte Response, Regulates Expression of Costimulatory Molecules on Antigen-Presenting Cells, and Rescues Pregnancy in Mouse Pregnancy Loss Model.

The loss of immune tolerance to fetal antigens may result in reproductive failure. The downregulated number and activity of T regulatory lymphocytes, which are critical for the establishment of immune tolerance to fetal antigens, during pregnancy may lead to miscarriage. The adoptive transfer of Tregs prevents fetal loss in abortion-prone mice. Recently, we demonstrated that the administration of tregitopes, which are short peptides found in human and mouse immunoglobulins (IgGs), decreased the incidence of abortions in female CBA/J mice mated with DBA/2J mice. Here, two non-IgG source peptides (SGS and LKD) that can potentially bind to the major histocompatibility complex II (MHC II) with high affinity and induce Treg expansion were designed in silico. The immune dysregulation-induced pregnancy failure mouse model was used to evaluate the effect of SGS and LKD on immune response and pregnancy outcome. The fetal death rate in the SGS-treated group was lower than that in the phosphate-buffered saline-treated group. SGS and LKD upregulated the splenic pool of Tregs and modulated the T-helper cell (Th1)/Th2-related cytokine response at the preimplantation stage. Additionally, SGS and LKD downregulated the expression of CD80 and MHC class II molecules in splenic CD11c+ antigen-presenting cells. Thus, SGS treatment can result in beneficial pregnancy outcomes. Additionally, SGS peptide-mediated immunomodulation can be a potential therapeutic strategy for immune dysregulation-induced pregnancy failure.

Knockdown of Lrp1 in RAW264 cells inhibits osteoclast differentiation and osteoclast-osteoblast interactions in vitro.

Low density lipoprotein receptor-related protein 1 (LRP1), a multifunctional cell surface protein, is expressed in bone marrow-derived macrophages. While LRP1 is thought to be a suppressor of osteoclast differentiation at late stages, its function at early stages remains unclear. Here we demonstrate that Lrp1 stable knockdown by lentiviral short hairpin RNA in macrophage cell line RAW264 cells inhibited RANKL-induced osteoclast formation and osteoclastic master transcription factor Nfatc1 mRNA expression as assessed by quantitative RT-PCR. Furthermore, knockdown of the Lrp1 gene suppressed not only differentiation, but also proliferation, and inhibitory effects on osteoblastic ALP activity by osteoclast-derived humoral factors. Thus, we propose that LRP1 in macrophages is required for both differentiation into osteoclasts and osteoclast-osteoblast interactions.

Bright expression of CD91 identifies highly activated human dendritic cells that can be expanded by defensins.

CD91 is a scavenger receptor expressed by different immune cells and its ligands defensins have been demonstrated to contribute to immune responses against infections and tumours. We previously demonstrated that CD91 is expressed on human monocyte-derived dendritic cells (moDCs) and that human defensins stimulate in vitro the activation of these cells. In this study, we observed that CD91 is expressed at different levels on two distinct moDC subsets: CD91(dim) and CD91(bright) moDCs. Although CD91(bright) moDCs represented a small proportion of total moDCs, this subset showed higher levels of activation and maturation markers compared with CD91(dim) moDCs. The frequency of CD91(bright) moDCs increased by ~ 50% after in vitro stimulation with recombinant human neutrophil peptide-1 (rHNP-1) and recombinant human ß defensin-1 (rHBD-1), while lipopolysaccharide (LPS) stimulation decreased it by ~ 35%. Both defensins up-regulated moDC expression of CD80, CD40, CD83 and HLA-DR, although to a lower extent compared with LPS. Notably, upon culture with rHNP-1 and rHBD-1, CD91(bright) moDCs maintained their higher activation/maturation status, whereas this was lost upon culture with LPS. Our findings suggest that defensins promote the differentiation into activated CD91(bright) DCs and may encourage the exploitation of the CD91/defensins axis as a novel therapeutic strategy to potentiate antimicrobial and anti-tumour immune response.

Development of a Humanized Antibody Targeting Extracellular HSP90α to Suppress Endothelial-Mesenchymal Transition-Enhanced Tumor Growth of Pancreatic Adenocarcinoma Cells.

Extracellular HSP90α (eHSP90α) is a promoter of tumor development and malignant progression. Patients with malignancies, including pancreatic ductal adenocarcinoma (PDAC), have generally shown 5~10-fold increases in serum/plasma eHSP90α levels. In this study, we developed a humanized antibody HH01 to target eHSP90α and evaluated its anticancer efficacy. HH01, with novel complementarity-determining regions, exhibits high binding affinity toward HSP90α. It recognizes HSP90α epitope sites 235AEEKEDKEEE244 and 251ESEDKPEIED260, with critical amino acid residues E237, E239, D240, K241, E253, and K255. HH01 effectively suppressed eHSP90α-induced invasive and spheroid-forming activities of colorectal cancer and PDAC cell lines by blocking eHSP90α's ligation with the cell-surface receptor CD91. In mouse models, HH01 potently inhibited the tumor growth of PDAC cell grafts/xenografts promoted by endothelial-mesenchymal transition-derived cancer-associated fibroblasts while also reducing serum eHSP90α levels, reflecting its anticancer efficacy. HH01 also modulated tumor immunity by reducing M2 macrophages and reinvigorating immune T-cells. Additionally, HH01 showed low aggregation propensity, high water solubility, and a half-life time of >18 days in mouse blood. It was not cytotoxic to retinal pigmented epithelial cells and showed no obvious toxicity in mouse organs. Our data suggest that targeting eHSP90α with HH01 antibody can be a promising novel strategy for PDAC therapy.

Multi-faceted role of LRP1 in the immune system.

Graft versus host disease (GVHD) represents the major complication after allogeneic hematopoietic stem cell transplantation (Allo-SCT). GVHD-prone patients rely on GVHD prophylaxis (e.g. methotrexate) and generalized anti-GVHD medical regimen (glucocorticoids). New anti-GVHD therapy strategies are being constantly explored, however there is an urgent need to improve current treatment, since GVHD-related mortality reaches 22% within 5 years in patients with chronic GVHD. This review is an attempt to describe a very well-known receptor in lipoprotein studies - the low-density lipoprotein receptor related protein 1 (LRP1) - in a new light, as a potential therapeutic target for GVHD prevention and treatment. Our preliminary studies demonstrated that LRP1 deletion in donor murine T cells results in significantly lower GVHD-related mortality in recipient mice with MHC (major histocompatibility complex) -mismatched HSCT. Given the importance of T cells in the development of GVHD, there is a significant gap in scientific literature regarding LRP1's role in T cell biology. Furthermore, there is limited research interest and publications on this classical receptor molecule in other immune cell types. Herein, we endeavor to summarize existing knowledge about LRP1's role in various immune cells to demonstrate the possibility of this receptor to serve as a novel target for anti-GVHD treatment.

Extracellular HMGB1 Impairs Macrophage-Mediated Efferocytosis by Suppressing the Rab43-Controlled Cell Surface Transport of CD91.

High-mobility group box 1 (HMGB1) protein can impair phagocyte function by suppressing the macrophage-mediated clearance of apoptotic cells (ACs), thereby delaying inflammation resolution in the lungs and allowing the progression of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the precise mechanism underlying this HMGB1-mediated inhibition of efferocytosis remains unknown. The aim of this study was to determine the effect of HMGB1 on macrophage-mediated efferocytosis. We discovered that HMGB1 prevented efferocytosis by bone marrow-derived macrophages (BMDMs) and suppressed the expression of Ras-related GTP-binding protein 43 (Rab43), a member of the Ras-associated binding (Rab) family. The downregulation of Rab43 expression resulted in impaired clearance of apoptotic thymocytes by BMDMs. Subsequent analysis of HMGB1-treated and Rab43-deficient BMDMs revealed the inhibited transport of cluster of differentiation 91 (CD91), a phagocyte recognition receptor, from the cytoplasm to the cell surface. Notably, Rab43 directly interacted with CD91 to mediate its intercellular trafficking. Furthermore, Rab43 knockout delayed the inflammation resolution and aggravated the lung tissue damage in mice with ALI. Therefore, our results provide evidence that HMGB1 impairs macrophage-mediated efferocytosis and delays inflammation resolution by suppressing the Rab43-regulated anterograde transport of CD91, suggesting that the restoration of Rab43 levels is a promising strategy for attenuating ALI and ARDS in humans.

YO2 Induces Melanoma Cell Apoptosis through p53-Mediated LRP1 Downregulation.

The multifunctional endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP1) has been implicated in melanoma growth. However, the mechanism of LRP1 expression in melanoma cells remains only partially understood. In most melanomas, the TP53 tumor suppressor is retained as a non-mutated, inactive form that fails to suppress tumors. We identify TP53 as a regulator of LRP1-mediated tumor growth. TP53 enhances the expression of miRNA miR-103/107. These miRNAs target LRP1 expression on melanoma cells. TP53 overexpression in human and murine melanoma cells was achieved using lentivirus or treatment with the small molecule YO-2, a plasmin inhibitor known to induce apoptosis in various cancer cell lines. TP53 restoration enhanced the expression of the tumor suppressor miR-103/107, resulting in the downregulation of LRP1 and suppression of tumor growth in vivo and in vitro. Furthermore, LRP1 overexpression or p53 downregulation prevented YO-2-mediated melanoma growth inhibition. We identified YO-2 as a novel p53 inducer in melanoma cells. Cotreatment of YO-2 with doxorubicin blocked tumor growth in vivo and in a murine melanoma model, suggesting that YO-2 exerts anti-melanoma effects alone or in combination with conventional myelosuppressive drugs.

Extracellular HSP90α Induces MyD88-IRAK Complex-Associated IKKα/ß-NF-κB/IRF3 and JAK2/TYK2-STAT-3 Signaling in Macrophages for Tumor-Promoting M2-Polarization.

M2-polarization and the tumoricidal to tumor-promoting transition are commonly observed with tumor-infiltrating macrophages after interplay with cancer cells or/and other stroma cells. Our previous study indicated that macrophage M2-polarization can be induced by extracellular HSP90α (eHSP90α) secreted from endothelial-to-mesenchymal transition-derived cancer-associated fibroblasts. To extend the finding, we herein validated that eHSP90α-induced M2-polarized macrophages exhibited a tumor-promoting activity and the promoted tumor tissues had significant increases in microvascular density but decreases in CD4+ T-cell level. We further investigated the signaling pathways occurring in eHSP90α-stimulated macrophages. When macrophages were exposed to eHSP90α, CD91 and toll-like receptor 4 (TLR4) functioned as the receptor/co-receptor for eHSP90α binding to recruit interleukin (IL)-1 receptor-associated kinases (IRAKs) and myeloid differentiation factor 88 (MyD88), and next elicited a canonical CD91/MyD88-IRAK1/4-IκB kinase α/ß (IKKα/ß)-nuclear factor-κB (NF-κB)/interferon regulatory factor 3 (IRF3) signaling pathway. Despite TLR4-MyD88 complex-associated activations of IKKα/ß, NF-κB and IRF3 being well-known as involved in macrophage M1-activation, our results demonstrated that the CD91-TLR4-MyD88 complex-associated IRAK1/4-IKKα/ß-NF-κB/IRF3 pathway was not only directly involved in M2-associated CD163, CD204, and IL-10 gene expressions but also required for downregulation of M1 inflammatory cytokines. Additionally, Janus kinase 2 (JAK2) and tyrosine kinase 2 (TYK2) were recruited onto MyD88 to induce the phosphorylation and activation of the transcription factor signal transducer and activator of transcription-3 (STAT-3). The JAK2/TYK2-STAT-3 signaling is known to associate with tumor promotion. In this study, the MyD88-JAK2/TYK2-STAT-3 pathway was demonstrated to contribute to eHSP90α-induced macrophage M2-polarization by regulating the expressions of M1- and M2-related genes, proangiogenic protein vascular endothelial growth factor, and phagocytosis-interfering factor Sec22b.

Complement C1q Interacts With LRP1 Clusters II and IV Through a Site Close but Different From the Binding Site of Its C1r and C1s-Associated Proteases.

LRP1 is a large endocytic modular receptor that plays a crucial role in the scavenging of apoptotic material through binding to pattern-recognition molecules. It is a membrane anchored receptor of the LDL receptor family with 4 extracellular clusters of ligand binding modules called cysteine rich complement-type repeats that are involved in the interaction of LRP1 with its numerous ligands. Complement C1q was shown to interact with LRP1 and to be implicated in the phagocytosis of apoptotic cells. The present work aimed at exploring how these two large molecules interact at the molecular level using a dissection strategy. For that purpose, recombinant LRP1 clusters II, III and IV were produced in mammalian HEK293F cells and their binding properties were investigated. Clusters II and IV were found to interact specifically and efficiently with C1q with K Ds in the nanomolar range. The use of truncated C1q fragments and recombinant mutated C1q allowed to localize more precisely the binding site for LRP1 on the collagen-like regions of C1q (CLRs), nearby the site that is implicated in the interaction with the cognate protease tetramer C1r2s2. This site could be a common anchorage for other ligands of C1q CLRs such as sulfated proteoglycans and Complement receptor type 1. The use of a cellular model, consisting in CHO LRP1-null cells transfected with full-length LRP1 or a cluster IV minireceptor (mini IV) confirmed that mini IV interacts with C1q at the cell membrane as well as full-length LRP1. Further cellular interaction studies finally highlighted that mini IV can endorse the full-length LRP1 binding efficiency for apoptotic cells and that C1q has no impact on this interaction.

Deferoxamine therapy reduces brain hemin accumulation after intracerebral hemorrhage in piglets.

Hemopexin (Hpx) is critical for hemin scavenging after the erythrocyte lysis that occurs following intracerebral hemorrhage (ICH). Low-density lipoprotein receptor-related protein-1 (LRP1, also called CD91) is an important receptor through which the hemin-Hpx complex can undergo endocytosis. This study investigated changes in the hemin-Hpx-CD91 axis in both hematoma and perihematomal tissue in a large animal ICH model. The effect of deferoxamine (DFX) on hemin-Hpx-CD91 was also examined. The study consisted of two parts. First, piglets had an injection of autologous blood into the right frontal lobe of brain and were euthanized from day 1 to day 7. Hematoma and perihematomal tissue of brains were used for hemin assay, immunohistochemistry, and immunofluorescence. Second, piglets with ICH were treated with deferoxamine or vehicle, and were euthanized for hemin measurement and Hpx and CD91 immunohistochemistry. We found that there was an increase of hemin levels within the hematoma and perihematomal brain tissue after ICH. Hpx and CD91-positive cells were present in the clot and perihematomal tissue from day 1. Hpx and CD91 positive cells were Iba1 positive. After DFX therapy, hemin dropped markedly in the hematoma and perihematomal brain tissue. Furthermore, DFX treatment decreased the number of Hpx and CD91 positive cells in and around the hematoma. In conclusion, hemin accumulation occurs in and around the hematoma. Increases in Hpx and CD91 may be important in scavenging that hemin. DFX treatment decreased hemin release from the hematoma and reduced the expression of Hpx and CD91.

Capsaicin-mediated apoptosis of human bladder cancer cells activates dendritic cells via CD91.

OBJECTIVES: Immunostimulation by anticancer cytotoxic drugs is needed for long-term therapeutic success. Activation of dendritic cells (DCs) is crucial to obtain effective and long-lasting anticancer T-cell mediated immunity. The aim of this study was to explore the effect of capsaicin-mediated cell death of bladder cancer cells on the activation of human monocyte-derived CD1a+ immature DCs. METHODS: Immature DCs (generated from human peripheral blood-derived CD14+ monocytes cultured with granulocyte-macrophage colony stimulating factor and interleukin-4) were cocultured with capsaicin (CPS)-induced apoptotic bladder cancer cells. DC activation was investigated using immunofluorescence and flow cytometric analysis for key surface molecules. In some experiments, CD91 was silenced in immature DCs. RESULTS: We found that capsaicin-mediated cancer cell apoptosis upregulates CD86 and CD83 expression on DCs, indicating the induction of DC activation. Moreover, silencing of CD91 (a common receptor for damage-associated molecular patterns, such as calreticulin and heat-shock protein-90/70) in immature DCs led to the inhibition of DC activation. CONCLUSIONS: Our data show that CPS-mediated cancer cell apoptosis activates DCs via CD91, suggesting CPS as an attractive candidate for cancer therapy.

The heat shock protein-CD91 pathway mediates tumor immunosurveillance.

Tumor immunosurveillance can be readily observed in mice and humans. Here, we examine how T-cell responses are primed during tumorigenesis, a condition in which immunostimulatory antigens are extraordinarily scarce. We recently demonstrated that the HSP-CD91 pathway is indispensable for antigen cross-presentation, and thus immunosurveillance, in cancer.

Properties of human blood monocytes. I. CD91 expression and log orthogonal light scatter provide a robust method to identify monocytes that is more accurate than CD14 expression.

BACKGROUND: This study was designed to improve identification of human blood monocytes by using antibodies to molecules that occur consistently on all stages of monocyte development and differentiation. METHODS: We examined blood samples from 200 healthy adults without clinically diagnosed immunological abnormalities by flow cytometry (FCM) with multiple combinations of antibodies and with a hematology analyzer (Beckman LH750). RESULTS: CD91 (α2 -macroglobulin receptor) was expressed only by monocytes and to a consistent level among subjects [mean median fluorescence intensity (MFI) = 16.2 ± 3.2]. Notably, only 85.7 ± 5.82% of the CD91(+) monocytes expressed high levels of the classical monocyte marker CD14, with some CD91(+) CD16(+) cells having negligible CD14, indicating that substantial FCM under-counts will occur when monocytes are identified by high CD14. CD33 (receptor for sialyl conjugates) was co-expressed with CD91 on monocytes but CD33 expression varied by nearly ten-fold among subjects (mean MFI = 17.4 ± 7.7). In comparison to FCM analyses, the hematology analyzer systematically over-counted monocytes and eosinophils while lymphocyte and neutrophil differential values generally agreed with FCM methods. CONCLUSIONS: CD91 is a better marker to identify monocytes than CD14 or CD33. Furthermore, FCM (with anti-CD91) identifies monocytes better than a currently used clinical CBC instrument. Use of anti-CD91 together with anti-CD14 and anti-CD16 supports the identification of the diagnostically significant monocyte populations with variable expression of CD14 and CD16.

Properties of human blood monocytes. II. Monocytes from healthy adults are highly heterogeneous within and among individuals.

BACKGROUND: Human blood monocytes are known to include subsets defined by the expression of CD14 and CD16 but otherwise are often assumed to be relatively homogeneous. However, we had observed additional heterogeneity that led us to a more extensive examination of monocytes. METHODS: Blood samples from 200 healthy adults without known immunological abnormalities were examined by analysis with a hematology analyzer and by flow cytometry (FCM) to determine leukocyte differential counts, to identify subsets and to measure expression of monocyte-associated molecules. RESULTS: The estimated cell counts of monocytes, neutrophils, total lymphocytes, and T cells all varied to a similar extent, that is, ±30-35%. The fractions of monocyte subsets defined by CD14 and CD16 or by CD163 expression also varied among individuals. FCM examinations showed that all the monocyte-associated molecules that were examined varied in expression in this increasing order-CD244, CD4, CD38, CD91, CD11b, toll-like receptor 2 (TLR2), TIA-1, CD14 (on CD14(Br+) cells), CD86, CD80, HLA-DQ, CD33, and HLA-DR. CONCLUSIONS: Human blood monocytes are heterogeneous among healthy adults with respect to cell counts, subsets, and the levels of expression of monocyte-associated molecules. An increase in the "non-classical" (CD14(Lo/Neg) /CD16(+) ) monocyte subset or in the expression of CD11b or TLR2 have known diagnostic/prognostic implications. CD244 and CD4 have well-defined functions on lymphocytes but perform unknown activities on monocytes although their expression appears more narrowly controlled. Together, these data suggest that monocytes should be more extensively examined in both clinical and basic contexts.