Myeloid Progenitor Inhibitory Factor Research Articles

Neonatal immune signatures differ by sex regardless of neurodevelopmental disorder status: Macrophage migration inhibitory factor (MIF) alone reveals a sex by diagnosis interaction effect

Immune dysregulation, including aberrant peripheral cytokine/chemokine levels, is implicated in neurodevelopmental disorders (NDD) such as autism spectrum disorder (ASD). While the diagnosis of ASD is more common in males compared to females, sex effects in immune dysregulation related to neurodevelopment remain understudied. The aim of this exploratory study was to determine whether there are sex-specific effects in neonatal immune dysregulation with respect to an ASD or delayed development (DD) diagnosis. We utilized the data from the Early Markers for Autism study, a population based case-control study of prenatal and neonatal biomarkers of ASD. The immune profile of newborns later diagnosed with ASD (n = 482, 91 females), DD (n = 140, 61 females) and sex-matched general population controls (GP; n = 378, 67 females) were analyzed using neonatal bloodspots (NBS) via 42-plex multiplex assay. Multiple linear regression analysis was performed to identify whether sex was associated with differences in cytokine/chemokine levels of children with ASD, DD, and GP. A sex by diagnosis interaction effect was observed only for the chemokine macrophage migration inhibitory factor (MIF), with males displaying higher levels of NBS MIF than females in the GP control group (p = 0.02), but not in ASD (p = 0.52) or DD (p = 0.29) groups. We found that regardless of child diagnosis, newborn bloodspot eluates from females had a significantly higher concentration than males with the same diagnosis of the chemokines granulocyte chemotactic protein 2 (GCP-2; p < 0.0001), macrophage inflammatory protein 2-alpha (GROβ; p = 0.002), interferon-inducible t-cell alpha chemoattractant (I-TAC; p < 0.0001), stromal cell-derived factor 1 alpha and beta (SDF-1α-β; p = 0.03), innate inflammatory chemokines interferon-gamma induced protein 10 (IP-10; p = 0.02), macrophage inflammatory protein 1-alpha (MIP-1α; p = 0.02), and Th1-related pro-inflammatory cytokine interleukin-12 active heterodimer (IL-12p70; p = 0.002). In contrast, males had a higher concentration than females of secondary lymphoid-tissue chemokine (6CKINE; p = 0.02), monocyte chemotactic protein 1 (MCP-1; p = 0.005) and myeloid progenitor inhibitory factor 1 (MPIF-1; p = 0.03). Results were similar when analyses were restricted to NBS from DD and ASD further classified as ASD with intellectual disability (ID), ASD without ID, and DD (GCP-2, p = 0.007; I-TAC, p = 0.001; IP-10, p = 0.005; IL-12p70, p = 0.03 higher in females; MPIF-1, p = 0.03 higher in male). This study is the first to examine sex differences in neonatal cytokine/chemokine concentrations, and whether these differences are associated with neurodevelopmental outcomes. Results highlight the importance of considering sex as a critical factor in understanding the immune system as it relates to child development.

Myeloid Progenitor Inhibitory Factor-1 (CCL23) Inhibits Lung Leukocyte Recruitment in a Primate Cardiopulmonary Bypass-Induced Pulmonary Ischaemia Model.

Bone marrow (BM)-derived polymorphonuclear leukocytes (PMNs) and monocytes (MO) induced by cardiopulmonary bypass (CPB) are highly proteolytic and cause postoperative lung injury. Although CCL23/Myeloid progenitor inhibitory factor-1 is a human CC chemokine with potent suppressor effects on myeloid progenitor cells, invivo inhibitory effects on BM-derived leukocyte kinetics associated with CPB are unknown. Two-hour CPB was surgically performed in cynomolgus monkeys and BM-derived leukocytes kinetics were monitored postoperatively by flow cytometry with 5'-bromo-2'-deoxyuridine (BrdU) and cytokine ELISA. Monkeys were given CCL23 (n=5) or saline (control, n=5) intravenously daily for 3 days before BrdU labelling and peripheral blood/bronchoalveolar lavage fluid (BALF) timepoint sampling to reveal BrdU-labelled cells. Levels of cytokines, CD11b, and L-selectin were considered leukocytic activation markers. The CCL23 treatment significantly prolonged BM transit of leukocytes (PMNs, 118.4±11.7-95.5±4.1 hours [control]; MO, 91.6±5.0-62.0±3.0 hours [control]) and reduced their alveolar appearance. The BM pool size of MO was decreased by CCL23 but PMNs were unaffected. CD11b, L-selectin expression of PMNs and MO during CPB, and post-surgical increases of interleukin (IL)-6, IL-8, TNF-α, MCP-1, and PMN elastase in the BALF were not suppressed. CCL23 treatment slows turnover of PMN and MO progenitors in BM and suppresses their circulatory release and lung recruitment. CCL23 has inhibitory effects specifically on the CPB-induced BM response and could hold value for preventing CPB-induced lung injury.

Highly multiplexed proteomic assessment of human bone marrow in acute myeloid leukemia

AbstractAcute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by abnormal clonal proliferation of myeloid progenitor cells found predominantly within the bone marrow (BM) and blood. Recent studies suggest that genetic and phenotypic alterations in the BM microenvironment support leukemogenesis and allow leukemic cells to survive and evade chemotherapy-induced death. However, despite substantial evidence indicating the role of tumor–host interactions in AML pathogenesis, little is known about the complex microenvironment of the BM. To address this, we performed novel proteomic profiling of the noncellular compartment of the BM microenvironment in patients with AML (n = 10) and age- and sex-matched healthy control subjects (n = 10) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). We show that proteomic assessment of blood or RNA-sequencing of BM are suboptimal alternate screening strategies to determine the true proteomic composition of the extracellular soluble compartment of AML patient BM. Proteomic analysis revealed that 168 proteins significantly differed in abundance, with 91 upregulated and 77 downregulated in leukemic BM. A highly connected signaling network of cytokines and chemokines, including IL-8, was found to be the most prominent proteomic signature associated with AML in the BM microenvironment. We report the first description of significantly elevated levels of the myelosuppressive chemokine CCL23 (myeloid progenitor inhibitory factor-1) in both AML and myelodysplastic syndrome patients and perform functional experiments supportive of a role in the suppression of normal hematopoiesis. This unique paired RNA-sequencing and proteomics data set provides innovative mechanistic insights into AML and healthy aging and should serve as a useful public resource.

A Novel Proteomic Profiling of the Bone Marrow Microenvironment Reveals Elevated Levels of the Chemokine CCL23 Isoforms in Acute Myeloid Leukemia

The bone marrow (BM) microenvironment is increasingly recognized as an important contributor to acute myeloid leukemia (AML) pathogenesis. However, despite growing interest in characterizing different components and cellular architecture of the BM niche and their biological significance in leukemogenesis, the proteomic constitution of the BM extracellular compartment that distinguishes a leukemic niche from its normal counterpart has not yet been fully described. We therefore performed a quantitative, large-scale proteomic analysis of 1,305 human proteins of the non-cellular compartment of BM (plasma) samples from ten relapsed or refractory AML patients and from ten age- and sex-matched healthy donors (HDs) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). This screen identified a total of 168 differentially abundant proteins, of which 91 were significantly more and 77 proteins significantly less abundant in leukemic BM compared with healthy marrow (FC ≥ 1.5, FDR ≤ 0.05). Comparative analysis of BM plasma and peripheral blood (PB) serum samples from the same AML patients and HDs revealed 65 similarly regulated proteins (37 up-regulated vs. 28 down-regulated) and 1 differently regulated protein between the two compartments. Out of the total 168 proteins, 102 proteins were specifically dysregulated only in the BM compartment. TruSeq Stranded Total RNA-sequencing (Illumina) was also performed using paired-end 75bp sequencing on a HiSeq 3000. RNA was isolated from PAXgene BM RNA tubes (Qiagen) collected in parallel with samples for proteomic analysis. Results of analysis of differentially expressed transcripts only partially overlapped with those candidates identified from our validated proteomic approach, indicating that sequencing of RNA derived from cellular sources of BM may be a suboptimal screening strategy to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. In addition to several previously reported proteins, our proteomics screen discovered numerous aberrantly expressed proteins in leukemic marrow whose role in AML pathogenesis is currently unknown. Using pathway analysis, we identified sets of proteins enriched for specific biological pathways including RAS, ephrin, PDGF, PI3K/AKT, MAPK, Notch, TLR, JAK-STAT, NFκB, Rap1, and Tie2 signaling pathways. A systems biology analysis approach revealed the highly connected network of cytokines and chemokines as the most striking AML-associated proteomic alteration in the BM. We identified IL-8 as a differentially expressed and key central molecule of this network in AML, consistent with recent reports. Importantly, we also identified significantly elevated levels of CKβ8 and CKβ8-1, alternatively spliced isoforms of the myelosuppressive chemokine CCL23 also known as myeloid progenitor inhibitory factor 1 (MPIF-1) or CKβ8, in both leukemic marrow and PB serum samples (Figure 1). Given the critical importance of cytopenias, often disproportional to the degree of leukemic marrow involvement, in the morbidity and mortality of patients with myelodysplastic syndrome (MDS) and AML, we subsequently confirmed this striking finding by performing orthogonal validation in a larger cohort of MDS and AML patients using an ELISA-based immunoassay. This novel finding suggests the possibility that CCL23 may play a role in suppression of normal hematopoiesis in MDS and AML. In support of this hypothesis, we demonstrated in vitro myelosuppressive effects of CCL23 isoforms on colony formation by human CD34+ hematopoietic stem and progenitor cells (HSPCs) in an in vitro colony forming unit assay, resulting in an approximately 2.5-fold decrease in CFU-GM and an evident decrease in CFU-GEMM counts. In summary, our broad and quantitative proteomic dataset of extracellular factors present in leukemic and normal aging bone marrow has already provided novel mechanistic insights into AML pathogenesis and should serve, together with paired RNA-sequencing information, as a useful public resource for the research community. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Hourigan:SELLAS Life Sciences Group AG: Research Funding; Merck, Sharpe &amp; Dohme: Research Funding.

Lysyl oxidase inhibition via β-aminoproprionitrile hampers human umbilical vein endothelial cell angiogenesis and migration invitro.

Lysyl oxidase (LOX) is an enzyme that oxidizes lysine residues in collagens and elastin. It stabilizes or remodels the extracellular matrix and basement membrane of blood vessels. Current oncology studies have revealed that LOX is upregulated in invasive cancer cells and bolstered cell movement, and LOX was observed to promote the angiogenesis and migration of endothelial cells. In the present study, angiogenesis and migration were examined in human umbilical vein endothelial cells (HUVECs). Following cell treatment with 0.1–0.4 mM β-aminoproprionitrile (BAPN), a specific inhibitor of LOX, angiogenesis was analyzed with a fibrin gel in vitro angiogenesis assay kit and migration was examined via a Boyden Chamber assay. Angiogenesis-associated gene expression was investigated with a microarray assay and confirmed with reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results showed that HUVEC angiogenesis substantially increased in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and phorbol 12-myristate 13-acetate (PMA). In addition, LOX inhibition blocked the angiogenesis stimulated by VEGF bFGF and PMA, and the inhibition of LOX reduced the migration of HUVECs. Furthermore, the microarray and RT-qPCR revealed that BAPN downregulated myeloid progenitor inhibitory factor 1, and western blot analysis demonstrated that BAPN decreased the phosphorylation of MAPK and Akt, suggesting that the specific inhibitor of LOX, BAPN, may serve as an alternative strategy for preventing angiogenesis.

Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα.

CCL23, also known as myeloid progenitor inhibitory factor (MPIF)-1, macrophage inflammatory protein (MIP)-3, or CKβ8, is a member of the CC chemokine subfamily exerting its effects via CCR1 binding. By doing so, CCL23 selectively recruits resting T lymphocytes and monocytes, inhibits proliferation of myeloid progenitor cells and promotes angiogenesis. Previously, we and other groups have reported that human neutrophils are able to produce chemokines upon appropriate activation, including CCR1-binding CCL2, CCL3, and CCL4. Herein, we demonstrate that human neutrophils display the capacity to also express and release CCL23 when stimulated by R848 and, to a lesser extent, by other pro-inflammatory agonists, including LPS, Pam3CSK4, and TNFα. Notably, we show that, on a per cell basis, R848-activated neutrophils produce higher levels of CCL23 than autologous CD14+-monocytes activated under similar experimental conditions. By contrast, we found that, unlike CD14+-monocytes, neutrophils do not produce CCL23 in response to IL-4, thus indicating that they express CCL23 in a stimulus-specific fashion. Finally, we show that the production of CCL23 by R848-stimulated neutrophils is negatively modulated by IFNα, which instead enhances that of CCL2. Together, data extend our knowledge on the chemokines potentially produced by neutrophils. The ability of human neutrophils to produce CCL23 further supports the notion on the neutrophil capacity of orchestrating the recruitment of different cell types to the inflamed sites, in turn contributing to the control of the immune response.

CXCL10/IP-10 Is a Biomarker and Mediator for Kawasaki Disease

Kawasaki disease (KD), an acute febrile vasculitis, is the most common cause of acquired heart disease in childhood; however, diagnosing KD can be difficult. To identify unique proteomic biomarkers that can be used to facilitate earlier diagnosis of KD. We enrolled 214 children with fever and clinical features suggestive of KD. Of those, only 100 were diagnosed with KD. Their plasma samples were globally analyzed for cytokines, chemokines, and cell adhesion molecules using an unbiased, large-scale, quantitative protein array. This study was conducted in 3 stages: discovery, replication, and blinded validation. During the discovery phase (n [KD]=37; n [control]=20), the expression of interleukin-17F, sCD40L, E-selectin, CCL23 (myeloid progenitor inhibitory factor 1), and CXCL10 (IFN-γ-inducible protein 10 [IP-10]) were upregulated during the acute phase in patients with KD when compared with that in the controls. A notable increase was observed in the IP-10 levels (KD, 3037 ± 226.7 pg/mL; control, 672 ± 130.4 pg/mL; P=4.1 × 10(-11)). Receiver-operating characteristic analysis of the combined discovery and replication data (n [KD]=77; n [control]=77) showed that the IP-10 level had high area under the curve values (0.94 [95% confidence interval, 0.9055-0.9778]; sensitivity, 100%; and specificity, 77%). With 1318 pg/mL as the optimal cutoff, the blinded validation study confirmed that the IP-10 levels were a good predictor of KD. With intravenous immunoglobulin treatment, the IP-10 levels returned to normal. The downstream receptor of IP-10, CXCR3, was activated in the T cells of patients with acute KD. IP-10 may be used as a biomarker to facilitate KD diagnosis, and it may provide clues about the pathogenesis of KD.

Low Serum Levels of Myeloid Progenitor Inhibitory Factor-1 Predict Good Response to Methotrexate in Rheumatoid Arthritis

Background. Although the benchmark in the treatment of rheumatoid arthritis remains methotrexate, only 70% of patients respond. Thus, there is a need for predictive biomarkers. This study planned to evaluate serum levels of myeloid progenitor inhibitory factor-1 (MPIF-1) and monocyte chemoattractant protein 2 (MCP-2)—as biomarkers. Methods. Patients with rheumatoid arthritis (RA) having high disease activity (DAS28-3v ≥ 5.1) were treated with oral methotrexate (MTX) for 12 weeks. Disease activity was measured by DAS28-3v (Modified Disease Activity Score 3 variables). Serum samples were stored at baseline and 12 weeks. Results. This study included 46 patients (F : M = 35 : 11) having mean (±SD) age of 42.6 ± 11.3 yrs, disease duration of 4.7 ± 4.5 yrs, and DAS28-3v of 6.1 ± 0.8. Serum MPIF1 was elevated in patients compared to controls (1636.7 ± 1009.7, 441.2 ± 173.8 pg/mL, P < 0.001), but there was no difference in MCP2 levels (31.4 ± 11.9, 33.8 ± 24.0 pg/mL). Baseline MPIF-1 level was lower in good responders (ΔDAS28-3v ≥ 1.2, N = 9) compared to poor responders (ΔDAS28-3v < 0.6, N = 27) (1171.0 ± 670.8, 1816.7 ± 1154.1 pg/mL, P = 0.05). On ROC analysis, baseline MPIF1 performed reasonably to predict good response; that is, ΔDAS28-3v ≥ 1.2 (AUC 0.68, 95% CI 0.50–0.87). Conclusions. Lower baseline MPIF1 level predicted a good response to methotrexate at 12 weeks.

Prevalence of subclinical atherosclerosis is increased in systemic sclerosis and is associated with serum proteins: a cross-sectional, controlled study of carotid ultrasound

SSc is associated with an increased prevalence of atherosclerosis (ATS). This study assessed the prevalence of subclinical ATS as measured by carotid US and explored serum proteins to identify potential biomarkers of SSc-ATS. Forty-six SSc female patients and 46 age- and ethnicity-matched controls underwent carotid US to assess the presence of plaque and carotid intima media thickness (CIMT). Abstracted data included demographics, ATS risk factors and serum measurements [cholesterol, proinflammatory high-density lipoprotein (piHDL), CRP, lipoproteins]. Serum cytokines/proteins analyses included circulating type I IFN activity by quantifying IFN-inducible genes, soluble junctional adhesion molecule A (sJAM-A) and 100 serum proteins by using a microplate-based multiplex platform. Proteins significant at P < 0.05 on bivariate analyses for the presence of plaque were used to develop a composite measure. Patients with SSc had more plaque (45.6% vs 19.5%, P = 0.01) but similar CIMT compared with controls. Multiplex analysis detected significant associations between serum proteins of inflammation, vasculopathy and fibrosis with ATS in SSc, including IL-2, IL-6, CRP, keratinocyte growth factor, intercellular adhesion molecule 1, endoglin, plasminogen activator inhibitor 1 and insulin-like growth factor binding protein 3 associated with carotid plaque. Myeloid progenitor inhibitory factor 1, serum amyloid A, thrombomodulin, N-terminal pro-brain natriuretic peptide (BNP), and Clara cell secretory protein 16 kD correlated with CIMT. The median composite score for the plaque group was 6 and for the no plaque group it was 2 (P < 0.0001). Patients with SSc have a higher prevalence of carotid plaque than matched controls, and patients with SSc-plaque vs patients without plaque have elevated serum proteins implicated in both vasculopathy and fibrosis. Further studies are needed to evaluate the role of these proteins in SSc compared with healthy controls.

Potential involvement of CCL23 in atherosclerotic lesion formation/progression by the enhancement of chemotaxis, adhesion molecule expression, and MMP-2 release from monocytes

CCL23 [Ckβ8-1/myeloid progenitor inhibitory factor 1 (MPIF1)/macrophage inflammatory protein-3 (MIP3)], a member of the CC chemokine family, is involved in leukocyte trafficking, and implicated in inflammatory diseases. In the present study, we investigated the role of CCL23 in the development of human atherosclerosis, which is characterized by an inflammatory disease. CCL23 transcripts were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and CCL23 protein by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Expression of adhesion molecules was determined by flow cytometry, and matrix metalloproteinase-2 (MMP-2) levels by zymography. Proatherogenic factors such as oxidized low-density lipoprotein (oxLDL) and oxidative stress markedly enhanced CCL23 release from human THP-1 macrophages. CCL23 stimulated chemotaxis of human THP-1 monocytes in a dose-dependent manner and enhanced the expression of adhesion molecule CD11c, as well as release of MMP-2 from the THP-1 monocytes. Moreover, CCL23 expression at the mRNA level was significantly higher in human atherosclerotic lesions than in normal arteries, and CCL23 protein was co-expressed with CD68, a specific marker for macrophages. Circulating levels of plasma CCL23 were higher in atherosclerotic patients than in normal subjects. These findings suggest that CCL23 plays a role in the development of human atherosclerosis. CCL23 may be a useful target for the development of antiatherogenic agents.

Modeling Human Endometrial Decidualization from the Interaction between Proteome and Secretome

Decidualization of the human endometrium, which involves morphological and biochemical modifications of the endometrial stromal cells (ESCs), is a prerequisite for adequate trophoblast invasion and placenta formation. This study aims to investigate the proteome and secretome of in vitro decidualized ESCs. These data were combined with published genomic information and integrated to model the human decidualization interactome. Prospective experimental case-control study. A private research foundation. Sixteen healthy volunteer ovum donors. Endometrial samples were obtained, and ESCs were isolated and decidualized in vitro. Two-dimensional difference in-gel electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, Western blot, human protein cytokine array, ELISA, and bioinformatics analysis were performed. The proteomic analysis revealed 60 differentially expressed proteins (36 over- and 24 underexpressed) in decidualized versus control ESCs, including known decidualization markers (cathepsin B) and new biomarkers (transglutaminase 2, peroxiredoxin 4, and the ACTB protein). In the secretomic analysis, a total of 13 secreted proteins (11 up- and 2 down-regulated) were identified, including well-recognized markers (IGF binding protein-1 and prolactin) and novel ones (myeloid progenitor inhibitory factor-1 and platelet endothelial cell adhesion molecule-1). These proteome/secretome profiles have been integrated into a decidualization interactome model. Proteomic and secretomic have been used as hypothesis-free approaches together with complex bioinformatics to model the human decidual interactome for the first time. We confirm previous knowledge, describe new molecules, and we have built up a model for human in vitro decidualization as invaluable tool for the diagnosis, therapy, and interpretation of biological phenomena.

Differential chemokine activation of CC chemokine receptor 1‐regulated pathways: ligand selective activation of Gα 14‐coupled pathways

Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptor-ligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1-coupled pathways by known CCR1 agonists. Chemokines leukotactin-1, macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic peptide (MCP)-3, RANTES, and MIP-1delta all inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP-1delta was unable to signal via G14-, G16- or chimeric 16z44-coupled pathways. In a stable cell line expressing CCR1 and Galpha14, all of these five chemokines along with hemofiltrate CC chemokine (HCC)-1 and myeloid progenitor inhibitory factor (MPIF)-1 were able to stimulate G(i/o)-coupled pathways, but MIP-1delta, HCC-1 and MPIF-1 were unable to activate G14-mediated stimulation of phospholipase Cbeta activity. In addition, MIP-1delta was unable to promote the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase. This suggests that different chemokines are able to selectively activate CCR1-coupled pathways, probably because of different intrinsic ligand efficacies. CCR1 and Galpha14 or Galpha16 are co-expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine-tune intracellular signaling pathways.

Comprehensive Mapping of Poxvirus vCCI Chemokine-binding Protein: EXPANDED RANGE OF LIGAND INTERACTIONS AND UNUSUAL DISSOCIATION KINETICS

In compiling a comprehensive map of the ligand binding capacity of elements within the chemokine system, we have determined the spectrum of chemokines capable of interacting with the poxvirus-encoded viral CC chemokine inhibitor, vCCI. More than 80 chemokines were tested in parallel for their ability to displace radiolabeled signature chemokines from vCCI. Of these chemokines, 26 showed potential high affinity interactions. These interactions revealed an expanded spectrum of binding capacity for vCCI to now include molecules such as human myeloid progenitor inhibitory factor-1 as ligands. In addition, high affinity viral protein-protein interactions were revealed. For example, binding between poxvirus vCCI and the herpesvirus vMIP-II from HHV8 occurs with IC(50) approximately 10-50 nm. Unusual dissociation kinetics were observed between certain chemokines and vCCI. Notably, many ligands displayed a precipitous displacement profile, suggesting marked positive cooperativity of binding. Finally, heterologous competition provided evidence for overlapping but distinct binding sites for the many chemokines that bind to vCCI. The determination of the binding fingerprint and unusual binding interactions of vCCI with a large number of chemokines suggest a finely honed evolutionary strategy of chemokine sequestration during viral infection.

Changes in gene expression in macrophages infected with Mycobacterium tuberculosis: a combined transcriptomic and proteomic approach

SummaryWe investigated the changes which occur in gene expression in the human macrophage cell line, THP1, at 1, 6 and 12 hr following infection with Mycobacterium tuberculosis. The analysis was carried out at the transcriptome level, using microarrays consisting of 375 human genes generally thought to be involved in immunoregulation, and at the proteomic level, using two‐dimensional gel electrophoresis and mass spectrometry. The analysis of the transcriptome using microarrays revealed that many genes were up‐regulated at 6 and 12 hr. Most of these genes encoded proteins involved in cell migration and homing, including the chemokines interleukin (IL)‐8, osteopontin, monocyte chemotactic protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), regulated on activation, normal, T‐cell expressed and secreted (RANTES), MIP‐1β, MIP‐3α, myeloid progenitor inhibitory factor‐1 (MPIF‐1), pulmonary and activation regulated chemokine (PARC), growth regulated gene‐β (GRO‐β), GRO‐γ, MCP‐2, I‐309, and the T helper 2 (Th2) and eosinophil‐attracting chemokine, eotaxin. Other genes involved in cell migration which were up‐regulated included the matrix metalloproteinase MMP‐9, vascular endothelial growth factor (VEGF) and its receptor Flk‐1, the chemokine receptor CCR3, and the cell adhesion molecules vesicular cell adhesion molecule‐1 (VCAM‐1) and integrin a3. In addition to the chemokine response, genes encoding the proinflammatory cytokines IL‐1β (showing a 433‐fold induction), IL‐2 and tumour necrosis factor‐α (TNF‐α), were also found to be induced at 6 and/or 12 hr. It was more difficult to detect changes using the proteomic approach. Nevertheless, IL‐1β was again shown to be strongly up‐regulated. The enzyme manganese superoxide dismutase was also found to be strongly up‐regulated; this enzyme was found to be macrophage‐, rather than M. tuberculosis, derived. The heat‐shock protein hsp27 was found to be down‐regulated following infection. We also identified a mycobacterial protein, the product of the atpD gene (thought to be involved in the regulation of cytoplasmic pH) in the infected macrophage extracts.

Solution Structure and Dynamics of Myeloid Progenitor Inhibitory Factor-1 (MPIF-1), A Novel Monomeric CC Chemokine

MPIF-1, a CC chemokine, is a specific inhibitor of myeloid progenitor cells and is the most potent activator of monocytes. The solution structure of myeloid progenitor inhibitor factor-1 (MPIF-1) has been determined by NMR spectroscopy. The structure reveals that MPIF-1 is a monomer with a well defined core except for termini residues and adopts the chemokine fold of three beta-strands and an overlying alpha-helix. In addition to the four cysteines that characterize most chemokines, MPIF-1 has two additional cysteines that form a disulfide bond. The backbone dynamics indicate that the disulfide bonds and the adjacent residues that include the functionally important N-terminal and N-terminal loop residues show significant dynamics. MPIF-1 is a highly basic protein (pI >9), and the structure reveals distinct positively charged pockets that could be correlated to proteoglycan binding. MPIF-1 is processed from a longer proprotein at the N terminus and the latter is also functional though with reduced potency, and both proteins exist as monomers under a variety of solution conditions. MPIF-1 is therefore unique because longer proproteins of all other chemokines oligomerize in solution. The MPIF-1 structure should serve as a template for future functional studies that could lead to therapeutics for preventing chemotherapy-associated myelotoxicity.

ANALYSIS OF EOSINOPHILS AND MYELOID PROGENITOR RESPONSES TO MODIFIED FORMS OF MPIF-2

Myeloid progenitor inhibitory factor (MPIF)-2 is a β-chemokine with select and potent activities on eosinophils and myeloid progenitors. In the β-chemokine family, biological activity is modulated by differential processing of the amino-terminus. Here, for MPIF-2, we describe the biological activities of NH2-terminal deletion mutants and compare regions necessary for eosinophil and myeloid progenitor activities. Five MPIF-2 proteins with deletions at the amino-terminus were produced in Escherichia coli and assayed for calcium mobilization, chemotaxis and receptor binding activities on eosinophils, and for their ability to inhibit colony formation of human myeloid bone marrow progenitors. For eosinophils, deletion of the first two amino acids did not markedly alter activity, while subsequent truncations result in a complete loss of activity. One of the MPIF-2 mutants, MPIF-2 (P30-R99) was converted from an agonist to an antagonist of eotaxin, MPIF-2 and MCP-4 functional responses in eosinophil calcium flux and chemotaxis assays. Surprisingly, while displaying a complete loss of agonist activity toward eosinophils, MPIF-2 (P30-R99) retains ability to inhibit human bone marrow myeloid progenitor cell colony formation. In addition, processing at the amino terminus of MPIF-2 in vivo, may result in a chemokine with altered biological activities.

Molecular Cloning of a Novel Human CC Chemokine (Eotaxin-3) That Is a Functional Ligand of CC Chemokine Receptor 3

Previously, we mapped the novel CC chemokine myeloid progenitor inhibitory factor 2 (MPIF-2)/eotaxin-2 to chromosome 7q11.23 (Nomiyama, H., Osborne, L. R., Imai, T., Kusuda, J., Miura, R., Tsui, L.-C., and Yoshie, O. (1998) Genomics 49, 339-340). Since chemokine genes tend to be clustered, unknown chemokines may be present in the vicinity of those mapped to new chromosomal loci. Prompted by this hypothesis, we analyzed the genomic region containing the gene for MPIF-2/eotaxin-2 (SCYA24) and have identified a novel CC chemokine termed eotaxin-3. The genes for MPIF-2/eotaxin-2 (SCYA24) and eotaxin-3 (SCYA26) are localized within a region of approximately 40 kilobases. By Northern blot analysis, eotaxin-3 mRNA was constitutively expressed in the heart and ovary. We have generated recombinant eotaxin-3 in a baculovirus expression system. Eotaxin-3 induced transient calcium mobilization specifically in CC chemokine receptor 3 (CCR3)-expressing L1.2 cells with an EC(50) of 3 nM. Eotaxin-3 competed the binding of (125)I-eotaxin to CCR3-expressing L1.2 cells with an IC(50) of 13 nM. Eotaxin-3 was chemotactic for normal peripheral blood eosinophils and basophils at high concentrations. Collectively, eotaxin-3 is yet another functional ligand for CCR3. The potency of eotaxin-3 as a CCR3 ligand seems, however, to be approximately 10-fold less than that of eotaxin. Identification of eotaxin-3 will further promote our understanding of the control of eosinophil trafficking and other CCR3-mediated biological phenomena. The strategy used in this study may also be applicable to identification of other unknown chemokine genes.

Dendritic cells and MPIF-1: chemotactic activity and inhibition of endogenous chemokine production by IFN-gamma and CD40 ligation.

We have examined the biological activity of the CC chemokine myeloid progenitor inhibitory factor 1 (MPIF-1) on human dendritic cells. MPIF-1 has chemotactic activity on dendritic cells derived from either peripheral blood monocytes or cord blood CD34+ progenitors. However, chemokine treatment did not induce further cell activation or maturation. In addition, MPIF-1 is constitutively released by monocyte-derived dendritic cells but not macrophages or monocytes (resting or stimulated). The proinflammatory stimuli lipopolysaccharide and tumor necrosis factor alpha, which induced the release of monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and interleukin-8, did not affect MPIF-1 release. In contrast, CD40 ligation and interferon-gamma treatment, while stimulating the production of the other chemokines, caused a pronounced reduction of MPIF-1 transcript and protein release. Thus, in dendritic cells the regulation of the production and release of MPIF-1 is distinct in comparison to other CC and CXC chemokines.

Peptide Mapping and Disulfide Bond Analysis of Myeloid Progenitor Inhibitory Chemokine and Keratinocyte Growth Factor by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Peptide mapping and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) were conducted to characterize the human genome-based recombinant proteins. Accurate mass values for the deleted forms of the myeloid progenitor inhibitory factor chemokine (MPIF-1d23), and the keratinocyte growth factor (KGF-2d33) were measured as 8848.55 ± 0.25 and 16,175.87 ± 0.89 Da, respectively. The mass accuracy of delayed ion extraction MALDI-MS measurements was within 20 ppm of the cDNA predicted value. Reduction and alkylation of the chemokine showed the presence of six cysteine residues and three disulfide bonds. Additional confirmation of disulfide bonding among the cysteine residues of the chemokine was demonstrated by identifying the RP-HPLC separated tryptic and endoprotease Glu-C peptides. Three methionine residues of the chemokine were identified by MALDI-MS of its cyanogen bromide (CNBr) cleavage products. The KGF-2d33 growth factor, however, lacked the disulfide bonding between the two-cysteine residues and contained two free sulfhydryl groups. Direct analysis of the growth factor CNBr digest showed 7542.99, 4993.4, and 3107.7 Da peptides, which identified the methionine residues. Peptide mapping mass spectrometry indicated that host-specific posttranslational modifications had not influenced the gene expression work.

Characterization of the Signal Transduction Pathway Activated in Human Monocytes and Dendritic Cells by MPIF-1, a Specific Ligand for CC Chemokine Receptor 1

The receptor specificity and signal transduction pathway has been identified and characterized for a truncated form of myeloid progenitor inhibitory factor-1 (MPIF-1(24-99)). MPIF-1 binds specifically to sites, in particular CCR1, shared with macrophage inflammatory protein-1alpha (MIP-1alpha) on the surface of human monocytes and dendritic cells, as inferred by its ability to compete for [125I]MIP-1alpha, but not for [125I]MIP-1beta or [125I]monocyte chemotactic protein-1(MCP-1) binding to intact cells. Based on calcium flux, MPIF-1 is an agonist on CCR1-transfected HEK-293 cells, monocytes, and dendritic cells, but not on CCR5-, CCR8-, or CX3CR1-transfected cells. The inhibitory effect of guanosine 5'-O-(3-thio-triphosphate) (GTP-gammaS) or pertussis toxin pretreatment on MPIF-1 binding and calcium mobilization, respectively, indicates the involvement of G proteins in the interaction of MPIF-1 and its receptor(s). The increase in intracellular free calcium concentration following MPIF-1 treatment is mainly due to the influx of calcium from an extracellular pool. However, a portion of the intracellular free calcium concentration is derived from a phospholipase C inhibitor-sensitive intracellular pool. MPIF-1 induces a rapid dose-dependent release of [3H]arachidonic acid from monocytes that is dependent on extracellular calcium and is blocked by phospholipase A2 (PLA2) inhibitors. Furthermore, PLA2 activation is shown to be necessary for filamentous actin formation in monocytes. Thus, the MPIF-1 signal transduction pathway appears to include binding to CCR1; transduction by G proteins; effector function by phospholipase C, protein kinase C, calcium flux, and PLA2; and cytoskeletal remodeling.