Chemotaxis Of Mononuclear Cells Research Articles

Development and characterization of novel mouse monoclonal antibodies against chicken chemokine CC motif ligand 4

Chemokine (C-C motif) ligand (CCL) 4 is a CC chemokine subfamily member defined by the sequential positioning of conserved cysteine residues. Upon the binding of G-protein-coupled receptors on the cell surface, CCL4 mediates a diverse set of biological processes including chemotaxis, tumorigenesis, homeostasis and thymopoiesis. Although the physiological roles of mammalian CCL4s were elucidated >20 years ago, there is limited information on the biological activities of chicken CCL4 (chCCL4). In the present study, we developed and characterized mouse monoclonal antibodies (mAbs) against chCCL4 to characterize better the immunological properties of chCCL4. Out of initial screening of >400 clones, two mAbs detecting chCCL4, 1A12 and 15D9, were identified and characterized using western blotting and chCCL4-specific antigen-capture enzyme-linked immunosorbent assay, and their neutralizing activity was validated by chCCL4-induced peripheral blood mononuclear cell chemotaxis assay. Furthermore, the intracellular expression of chCCL4 in various chicken cells by immunocytochemistry and flow cytometry was confirmed using 1A12 and 15D9 mAbs. These results collectively indicate that 1A12 and 15D9 mAbs specifically detect chicken CCL4 and they will be valuable immune reagents for basic and applied studies in avian immunology.

STUDY OF CXCL12, CCR4, EGFR GENE EXPRESSION IN MIGRATING MYELOMONOBLASTIC LEUKEMIA CELLS BEFORE AND AFTER CHEMOTHERAPY

Аim. A study of CXCL12 effect on the migration of mononuclear cells isolated from healthy patients, from patients with myelomonoblastic leukemia before and after chemotherapy and the study of CCR4, EGFR and CXCL12 genes expression after exposure to CXCL12. Materials and methods. The chemotaxis of mononuclear cells (MNCs) of healthy donors and patients with myelomonoblastic leukemia was studied in a Boyden chamber, followed by isolation of RNA, reverse transcription and PCR-RV. Results. A significant increase in myelomonoblasic cell chemotaxis towards CXCL12 after chemotherapy was demonstrated, as well as a decrease in the expression of this chemokine in tumor cells before chemotherapy after exposure to CXCL12. Сonclusion. Presumably, the tumor cells themselves produce CXCL12 in large amounts, which is necessary for the disturbance of intercellular interactions and further intravasation, whose production may decrease with external stimulation by the same chemokine. CXCL12 also helps to increase the expression level of EGFR and CCR4, which leads to increased tumor proliferation and migration of tumor cells.

BMP-2 induces human mononuclear cell chemotaxis and adhesion and modulates monocyte-to-macrophage differentiation.

Type 2 diabetes mellitus (T2DM) is a cardiovascular risk factor which leads to atherosclerosis, an inflammatory disease characterized by the infiltration of mononuclear cells in the vessel. Bone morphogenetic protein (BMP)‐2 is a cytokine which has been recently shown to be elevated in atherosclerosis and T2DM and to contribute to vascular inflammation. However, the role of BMP‐2 in the regulation of mononuclear cell function remains to be established. Herein, we demonstrate that BMP‐2 induced human monocyte chemotaxis via phosphoinositide 3 kinase and mitogen‐activated protein kinases. Inhibition of endogenous BMP‐2 signalling, by Noggin or a BMP receptor inhibitor, interfered with monocyte migration. Although BMP‐2 expression was increased in monocytes from T2DM patients, it could still stimulate their migration. Furthermore, BMP‐2 interfered with their differentiation into M2 macrophages. Finally, BMP‐2 both induced the adhesion of monocytes to fibronectin and endothelial cells (ECs), and promoted the adhesive properties of ECs, by increasing expression of adhesion and pro‐inflammatory molecules. Our data demonstrate that BMP‐2 could exert its pro‐inflammatory effects by inducing monocyte migration and adhesiveness to ECs and by interfering with the monocyte differentiation into M2 macrophages. Our findings provide novel insights into the mechanisms by which BMP‐2 may contribute to the development of atherosclerosis.

Truncation of CXCL12 by CD26 reduces its CXC chemokine receptor 4- and atypical chemokine receptor 3-dependent activity on endothelial cells and lymphocytes

The chemokine CXCL12 or stromal cell-derived factor 1/SDF-1 attracts hematopoietic progenitor cells and mature leukocytes through the G protein-coupled CXC chemokine receptor 4 (CXCR4). In addition, it interacts with atypical chemokine receptor 3 (ACKR3 or CXCR7) and glycosaminoglycans. CXCL12 activity is regulated through posttranslational cleavage by CD26/dipeptidyl peptidase 4 that removes two NH2-terminal amino acids. CD26-truncated CXCL12 does not induce calcium signaling or chemotaxis of mononuclear cells. CXCL12(3–68) was chemically synthesized de novo for detailed biological characterization. Compared to unmodified CXCL12, CXCL12(3–68) was no longer able to signal through CXCR4 via inositol trisphosphate (IP3), Akt or extracellular signal-regulated kinases 1 and 2 (ERK1/2). Interestingly, the recruitment of β-arrestin 2 to the cell membrane via CXCR4 by CXCL12(3–68) was abolished, whereas a weakened but significant β-arrestin recruitment remained via ACKR3. CXCL12-induced endothelial cell migration and signal transduction was completely abrogated by CD26. Intact CXCL12 hardly induced lymphocyte migration upon intra-articular injection in mice. In contrast, oral treatment of mice with the CD26 inhibitor sitagliptin reduced CD26 activity and CXCL12 cleavage in blood plasma. The potential of CXCL12 to induce intra-articular lymphocyte infiltration was significantly increased in sitagliptin-treated mice and CXCL12(3–68) failed to induce migration under both CD26-inhibiting and non-inhibiting conditions. In conclusion, CD26-cleavage skews CXCL12 towards β-arrestin dependent recruitment through ACKR3 and destroys the CXCR4-mediated lymphocyte chemoattractant properties of CXCL12 in vivo. Hence, pharmacological CD26-blockade in tissues may enhance CXCL12-induced inflammation.

Senescent vascular smooth muscle cells drive inflammation through an interleukin-1α-dependent senescence-associated secretory phenotype

Objective— Vascular smooth muscle cells (VSMCs) that become senescent are both present within atherosclerotic plaques and thought to be important to the disease process. However, senescent VSMCs are generally considered to only contribute through inaction, with failure to proliferate resulting in VSMC- and collagen-poor unstable fibrous caps. Whether senescent VSMCs can actively contribute to atherogenic processes, such as inflammation, is unknown. Approach and Results— We find that senescent human VSMCs develop a proinflammatory state known as a senescence-associated secretory phenotype. Senescent human VSMCs release high levels of multiple cytokines and chemokines driven by secreted interleukin-1α acting in an autocrine manner. Consequently, the VSMC senescence-associated secretory phenotype promotes chemotaxis of mononuclear cells in vitro and in vivo. In addition, senescent VSMCs release active matrix metalloproteinase-9, secrete less collagen, upregulate multiple inflammasome components, and prime adjacent endothelial cells and VSMCs to a proadhesive and proinflammatory state. Importantly, maintaining the senescence-associated secretory phenotype places a large metabolic burden on senescent VSMCs, such that they can be selectively killed by inhibiting glucose utilization. Conclusions— Senescent VSMCs may actively contribute toward the chronic inflammation associated with atherosclerosis through the interleukin-1α–driven senescence-associated secretory phenotype and the priming of adjacent cells to a proatherosclerotic state. These data also suggest that inhibition of this potentially important source of chronic inflammation in atherosclerosis requires blockade of interleukin-1α and not interleukin-1β. # Significance {#article-title-39}

Senescent Vascular Smooth Muscle Cells Drive Inflammation Through an Interleukin-1α-Dependent Senescence-Associated Secretory Phenotype.

Vascular smooth muscle cells (VSMCs) that become senescent are both present within atherosclerotic plaques and thought to be important to the disease process. However, senescent VSMCs are generally considered to only contribute through inaction, with failure to proliferate resulting in VSMC- and collagen-poor unstable fibrous caps. Whether senescent VSMCs can actively contribute to atherogenic processes, such as inflammation, is unknown. We find that senescent human VSMCs develop a proinflammatory state known as a senescence-associated secretory phenotype. Senescent human VSMCs release high levels of multiple cytokines and chemokines driven by secreted interleukin-1α acting in an autocrine manner. Consequently, the VSMC senescence-associated secretory phenotype promotes chemotaxis of mononuclear cells in vitro and in vivo. In addition, senescent VSMCs release active matrix metalloproteinase-9, secrete less collagen, upregulate multiple inflammasome components, and prime adjacent endothelial cells and VSMCs to a proadhesive and proinflammatory state. Importantly, maintaining the senescence-associated secretory phenotype places a large metabolic burden on senescent VSMCs, such that they can be selectively killed by inhibiting glucose utilization. Senescent VSMCs may actively contribute toward the chronic inflammation associated with atherosclerosis through the interleukin-1α-driven senescence-associated secretory phenotype and the priming of adjacent cells to a proatherosclerotic state. These data also suggest that inhibition of this potentially important source of chronic inflammation in atherosclerosis requires blockade of interleukin-1α and not interleukin-1β.

The therapeutic potential of a C-X-C chemokine receptor type 4 (CXCR-4) antagonist on hypertrophic scarring in vivo.

Effective prevention and treatment of hypertrophic scars (HTSs), a dermal form of fibrosis that frequently occurs following thermal injury to deep dermis, are unsolved significant clinical problems. Previously, we have found that stromal cell-derived factor 1/CXCR4 signaling is up-regulated during wound healing in burn patients and HTS tissue after thermal injury. We hypothesize that blood-borne mononuclear cells are recruited into wound sites after burn injury through the chemokine pathway of stromal cell-derived factor 1 and its receptor CXCR4. Deep dermal injuries to the skin are often accompanied by prolonged inflammation, which leads to chemotaxis of mononuclear cells into the wounds by chemokine signaling where fibroblast activation occurs and ultimately HTS are formed. Blocking mononuclear cell recruitment and fibroblast activation, CXCR4 antagonism is expected to reduce or minimize scar formation. In this study, the inhibitory effect of CXCR4 antagonist CTCE-9908 on dermal fibrosis was determined in vivo using a human HTS-like nude mouse model, in which split-thickness human skin is transplanted into full-thickness dorsal excisional wounds in athymic mice, where these wounds subsequently develop fibrotic scars that resemble human HTS as previously described. CTCE-9908 significantly attenuated scar formation and contraction, reduced the accumulation of macrophages and myofibroblasts, enhanced the remodeling of collagen fibers, and down-regulated the gene and protein expression of fibrotic growth factors in the human skin tissues. These findings support the potential therapeutic value of CXCR4 antagonist in dermal fibrosis and possibly other fibroproliferative disorders.

C-C motif chemokine CCL3 and canonical neutrophil attractants promote neutrophil extravasation through common and distinct mechanisms

Initial observations suggested that C-C motif chemokines exclusively mediate chemotaxis of mononuclear cells. In addition, recent studies also implicated these chemotactic cytokines in the recruitment of neutrophils. The underlying mechanisms remained largely unknown. Using in vivo microscopy on the mouse cremaster muscle, intravascular adherence and subsequent paracellular transmigration of neutrophils elicited by the chemokine (C-C motif) ligand 3 (CCL3, synonym MIP-1α) were significantly diminished in mice with a deficiency of the chemokine (C-C motif) receptor 1 (Ccr1(-/-)) or 5 (Ccr5(-/-)). Using cell-transfer techniques, neutrophil responses required leukocyte CCR1 and nonleukocyte CCR5. Furthermore, neutrophil extravasation elicited by CCL3 was almost completely abolished on inhibition of G protein-receptor coupling and PI3Kγ-dependent signaling, while neutrophil recruitment induced by the canonical neutrophil attractants chemokine (C-X-C motif) ligand 1 (CXCL1, synonym KC) or the lipid mediator platetelet-activating factor (PAF) was only partially reduced. Moreover, Ab blockade of β(2) integrins, of α(4) integrins, or of their putative counter receptors ICAM-1 and VCAM-1 significantly attenuated CCL3-, CXCL1-, or PAF-elicited intravascular adherence and paracellular transmigration of neutrophils. These data indicate that the C-C motif chemokine CCL3 and canonical neutrophil attractants exhibit both common and distinct mechanisms for the regulation of intravascular adherence and transmigration of neutrophils.

CC chemokine ligand-2 synergizes with the nonchemokine G protein-coupled receptor ligand fMLP in monocyte chemotaxis, and it cooperates with the TLR ligand LPS via induction of CXCL8

During inflammatory reactions, endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmitters to regulate host immune responses. These signaling circuitries are even more interfaced during infections, when microbial agonists activate TLR, RLR, and NLR receptors. On the basis of the discovery of synergy between chemokines for neutrophil attraction, we extend here this phenomenon between the chemokine MCP-1/CCL2 and the GPCR ligand fMLP or the TLR4 agonist LPS on monocytes. In fact, the bacterial tripeptide fMLP, but not the cytokines IL-1beta or IFN-gamma, significantly and dose-dependently synergized with CCL2 in monocyte chemotaxis. Furthermore, LPS rapidly induced the expression of IL-8/CXCL8 but not of the CCL2 receptor CCR2 in monocytic cells. In turn, the induced CXCL8 synergized with CCL2 for mononuclear cell chemotaxis, and the chemotactic effect was mediated by CXCR1/CXCR2, because CXCL8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to CCL2 intact. These data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults.

Activation of Human Valve Interstitial Cells by a Viridians Streptococci Modulin Induces Chemotaxis of Mononuclear Cells

Infective endocarditis is characterized by inflammatory infiltrates of mononuclear cells in infected cardiac valve leaflets. To delineate the role of valve interstitial cells (VICs) in leukocyte recruitment, we stimulated human VICs with glucosyltransferase, a modulin from viridians streptococci. Interstitial cells were activated directly by glucosyltransferase in a dose-dependent manner through concerted mitogen-activated protein kinase and nuclear factor-kappaB signaling pathways; activation resulted in up-regulation of synthesis and release of interleukin-6, interleukin-8, or monocyte chemoattractant protein-1 and enhanced transwell migration of U937 monocytic cells or primary mononuclear cells. The expression of glucosyltransferases and activation of VICs (nuclear localization of RelA) were detected in a rat model of experimental endocarditis. Proinflammatory cytokines also were detected in VICs from diseased human autopsy specimens but not in VICs from normal specimens. These results indicate that interstitial cells in the cardiac valve can be activated directly by bacterial modulins to recruit and retain mononuclear cells, likely contributing to the persistent inflammation characteristic of infective endocarditis.

Abstract 5554: Runx2 Represses TGFβ/Smad-Mediated Connective Tissue Growth Factor Gene Expression in Vascular Smooth Muscle Cells

[Background] Runx2, a key transcription factor for osteoblastic differentiation, is expressed in calcified atherosclerotic plaques. We recently reported that Runx2 represses vascular smooth muscle cells (VSMC) differentiation and promotes its osteogenic differentiation. Connective tissue growth factor (CTGF) has been implicated in the progression to vulnerable plaque by inducing mononuclear cell chemotaxis and VSMC apoptosis despite of its potent stimulatory effect on synthesis of extracellular matrix. In this study, we investigated the regulatory mechanism of CTGF gene expression by Runx2 in VSMC. [Methods and Results] RT-PCR analyses showed that adenovirally overexpressed Runx2 significantly repressed the basal expression of the CTGF gene in human aortic SMCs (HASMCs). Consistent with this, knockdown of the Runx2 expression in HASMCs by small interfering RNA (siRNA) increased CTGF mRNA levels. Luciferase assays showed that Runx2 reduced the transcriptional activity of the CTGF promoter. Transfection of a series of 5′-deletion constructs revealed that Runx2 inhibited CTGF expression through the sequence element located at 5′ untranslated region of CTGF mRNA. We next examined the effects of Runx2 on the TGFβ-induced CTGF expression. Runx2 overexpression significantly attenuated the TGFβ-mediated induction of CTGF expression in HASMCs, and knockdown of Runx2 by siRNA enhanced the induction of CTGF expression in response to TGFβ. Runx2 repressed TGFβ-induced CTGF promoter activity through the sequence containing Smad binding element (SBE), and luciferase assay using SBE-specific mutation construct showed that Runx2 repressed CTGF promoter activity in an SBE-dependent manner. Overexpression of Runx2 significantly reduced TGFβ and Smad3-mediated luciferase activity of 4xSBE-tkLuc, which contains four copies of SBE. Co-immunoprecipitation showed that Runx2 formed a complex with Smad4. [Conclusion] These data demonstrate that Runx2 represses basal and TGFβ-induced CTGF gene expression in VSMC, and thus suggest that besides the potential for inducing vascular calcification, Runx2 may affect plaque stability by modulating extracellular matrix synthesis through inhibiting CTGF gene expression and TGFβ signaling.

Accelerated Apoptosis and Aberrant Retention of Bone Marrow Cells in Myelokathexis Are Two Independent Pathways Triggered by Mutant CXCR4 and Normalized upon Treatment with Specific Inhibitors.

Myelokathexis (MK) is a rare congenital hematopoietic disease characterized by hypercellular marrow and severe neutropenia. In some patients there is an association of Warts, Hypogammaglobulinemia, and severe Infections with Myelokathexis (WHIM syndrome). We and others reported accelerated apoptosis of bone marrow myeloid cells in MK evidenced by electron microscopy and flow cytometry studies. The impaired cell survival in MK was associated with reduction in Bcl-X expression at least partially restored by G-CSF treatment. Heterozygous mutations in the CXCR4 gene were reported in most of the families with WHIM syndrome. Interaction of CXCR4, a G-protein coupled chemokine receptor with SDF-1 ligand plays a key role in homing and mobilization of CXCR4-expressing hematopoietic cells. It has been reported that expression of mutant CXCR4 leads to reduced receptor internalization, increased calcium flux and enhanced chemotaxis of transduced CD34+ cells toward SDF1. However, the mechanism of mutant CXCR4-triggered neutropenia in MK remains largely unknown. We examined 11 patients representing 6 unrelated families with MK and found heterozygous CXCR4 mutations in affected but not healthy family members. We identified truncation and deletion mutations including a novel mutation resulting in a deletion of the last 29 amino acids in the cytoplasmic domain of CXCR4. Expression of deletion and truncation mutants in human promyelocytic cells triggered apoptosis similar to that observed in MK patients. Specifically, the rate of apoptotic annexin-positive cells was approximately 2-fold higher in cells transfected with CXCR4 mutants compared with control cells transfected with normal CXCR4. Accelerated apoptosis appeared to stem from a significantly increased dissipation of mitochondrial membrane potential as determined by flow cytometry analysis of DIOC6-labeled cells expressing CXCR4 mutants compared with controls with normal CXCR4 (p<0.02). Similar to enhanced chemotaxis of mononuclear cells observed in MK patients, expression of CXCR4 mutants but not the wild type, resulted in a significant increase in directional motility of cells to SDF-1 (p<0.01). These data indicate that our cellular model appears to closely recapitulate the myelokathexis phenotype. Accelerated apoptosis, but not enhanced chemotaxis induced by mutant CXCR4 was reduced to near-normal level by zVAD-fmk-caspase-specific inhibitor. Interestingly, the mutant CXCR4-triggered increase in chemotaxis to SDF-1 was normalized by treatment with protein kinase C inhibitor. This reduction in mutant CXCR4-mediated increase in chemotaxis in response to the treatment with PKC inhibitor was similar to that observed in response to the treatment with AMD3100. Interestingly, combined treatment with both AMD3100 and PKC inhibitor did not result in a synergistic effect, suggesting that these agents may utilize the same signaling pathway. Of note, neither the treatment with PKC inhibitor not with AMD3100 affected accelerated apoptosis, suggesting that accelerated apoptosis and enhanced chemotaxis are two independent pathways triggered by mutant CXCR4. Importantly, treatment of myelokathexis patients' blood mononuclear cells with PKC inhibitor restored the abnormal chemotactic properties to near normal levels. Thus, our data suggest that AMD3100 and PKC inhibitor may be effective for treatment of myelokathexis patients with aberrant retention of hematopoietic cells in the bone marrow.

Abstract 311: Osteogenic Transcription Factor Runx2 Represses Connective Tissue Growth Factor Gene Expression And TGFβ Signaling In Vascular Smooth Muscle Cells

[Objective] Runx2, a key transcription factor in osteoblast differentiation, is expressed in calcified atherosclerotic plaques. We have recently shown that Runx2 represses vascular smooth muscle cells (VSMCs) differentiation and promotes their osteogenic differentiation. Connective tissue growth factor (CTGF) has been implicated in the progression to vulnerable plaque by inducing mononuclear cell chemotaxis and VSMCs apoptosis despite of its potent stimulatory effect on connective tissue cell the proliferation and extracellular matrix synthesis. To assess the role of Runx2 in the process of plaque development, we investigated the molecular mechanism of the CTGF gene expression by Runx2 in VSMCs. [Methods and Results] RT-PCR analyses showed that adenovirally overexpressed Runx2 significantly repressed the basal expression of the CTGF gene in human aortic SMCs (HASMCs). Consistent with this, knockdown of the Runx2 expression in HASMCs by small interfering RNA (siRNA) increased CTGF mRNA levels. Luciferase assays showed that Runx2 reduced the transcriptional activity of the CTGF promoter. Transfection of a series of 5′-deletion constructs revealed that Runx2 inhibited CTGF expression through the sequence element located at 5′ untranslated region of CTGF mRNA. We next examined the effects of Runx2 on the TGFβ-induced CTGF expression. Runx2 overexpression significantly repressed CTGF expression in HASMCs stimulated with TGFβ, and knockdown of Runx2 by siRNA enhanced the induction of CTGF expression in response to TGFβ. Runx2 repressed TGFβ-induced CTGF promoter activity through the sequence including Smad binding element (SBE). Overexpression of Runx2 significantly reduced TGFβ- and Smad3-mediated luciferase activity of Smad-dependent promoter which contains four copies of SBE. Biotinylated DNA pulldown assay using SBE of CTGF promoter showed that Runx2 formed a complex with Smad3 and Smad4. [Conclusion] Runx2 repressed basal and TGFβ-induced CTGF gene expression in VSMCs. Thus, in addition to the potential for inducing vascular calcification, Runx2 may affect plaque stability by modulating extracellular matrix synthesis through inhibiting CTGF gene expression and TGFβ signaling.

CXCL11 is involved in leucocyte recruitment to the central nervous system in neuroborreliosis

We investigated the role of the CXCR3 chemokine CXCL11 (I-TAC) for leukocyte recruitment to the CSF in neuroborreliosis (NB). CXCL11 levels in the CSF of 17 patients with acute NB were elevated compared with 20 non-inflammatory controls (100.1 vs. 54.1 pg/ml, p = 0.002). Using a modified Boyden chamber chemotaxis-assay, the CSF of patients with acute NB was more chemotactic than the control CSF-samples (p = 0.001). A strong correlation (p < 0.001) between CXCL11 levels, chemotactic activity and CSF-white cell count was detected. Though the chemotactic activity of CSF from NB patients was only partially reduced by a human recombinant CXCL11 antibody, these findings suggest that, among other chemotactic factors, CXCL11 may play a role in the chemotaxis of mononuclear cells in NB.

Connective Tissue Growth Factor Is Overexpressed in Complicated Atherosclerotic Plaques and Induces Mononuclear Cell Chemotaxis In Vitro

Atherosclerotic blood vessels overexpress connective tissue growth factor (CTGF) mRNA, but the role of CTGF in atherosclerosis remains controversial. To assess the hypothesis that CTGF is involved in atherosclerotic plaque progression, we investigated CTGF protein expression and distribution in the different types of plaque morphology. Serial cross-sections of 45 human carotid plaques were immunohistochemically analyzed for the presence of CTGF protein, neovascularization (von Willebrand factor), macrophages (CD68), and T cells (CD3). The lesions were categorized according to American Heart Association (AHA) classification as fibrous (type IV and V) or complicated plaques (type VI). The levels of CTGF were significantly higher in complicated compared with fibrous plaques (P=0.002). CTGF accumulated particularly in the rupture-prone plaque shoulder and in the areas of neovascularization or infiltration with inflammatory cells. Macrophage-like cells stained positive for CTGF protein in plaques. Subsequent in vitro studies showed that although monocyte-derived macrophages do not produce CTGF on stimulation with transforming growth factor-beta, lipopolysaccharide, or thrombin, they take it up from culture medium. Furthermore, CTGF induces mononuclear cell chemotaxis in a dose-dependent manner. CTGF protein is significantly increased in complicated compared with fibrous plaques and may enhance monocyte migration into atherosclerotic lesions, thus contributing to atherogenesis.

Vasoactive intestinal peptide impairs leucocyte migration but fails to modify experimental murine colitis

Inflammatory bowel diseases are chronic inflammatory disorders of the gastrointestinal tract. Vasoactive intestinal peptide (VIP) is a neuropeptide with known anti-inflammatory activity. We have demonstrated previously that administration of VIP inhibits leucocyte migration in a murine model of delayed-type hypersensitivity, and anti-inflammatory efficacy is supported by other studies. The aim of this study was to investigate the VIP effects in a murine model of intestinal inflammation. Colitis was induced in BALB/c mice by a 2.5 mg enema of 2,4,6-trinitrobenzenesulphonic acid (TNBS) and the mice were killed on day 7. Mice were administered either a 3-day (therapeutic) or 7-day (prophylactic) constant infusion of VIP by subcutaneously implanted mini-osmotic pumps, or intraperitoneal (i.p.) injection of VIP on alternate days over 7 days. Clinical disease scores, weight changes, histopathology of colon tissues, plasma VIP levels, cytokine levels and chemotaxis of peripheral blood mononuclear cells were evaluated. After administration of TNBS, mice quickly developed severe colitis accompanied by dramatic body weight loss (20% by day 6) and high mortality (30%). Prophylactic treatment using high-dose VIP abrogated leucocyte chemotaxis; however, it failed to ameliorate the weight loss and mortality. Moreover, VIP delivered either by constant infusion or i.p. failed to modify the clinical, histological or cytokine markers of disease. Our studies show that, despite an ability to inhibit chemokine-induced chemotaxis of mononuclear cells, VIP was unable to modulate TNBS-induced colitis. This contrasts with the efficacy of VIP in models of mild inflammatory disease and suggests that VIP is unlikely to provide a useful model for novel anti-IBD therapy.

Pollution and the immune response: atopic diseases--are we too dirty or too clean?

The effect of pollutants on the immune system has filled tomes and is almost always controversial whether the topic is disinfectants in drinking water or particulates and lung cancer. Even whether outdoor and indoor air quality is improving or declining has been a source of considerable investigation, consultation and often litigation. Few areas, however, yield so much uncertainty and debate as the role of airborne pollutants in atopy. Atopy is defined as an immunoglobulin E- (IgE) mediated allergic response to environmental antigens and can be manifested as rhinitis, asthma or atopic dermatitis. While there is some debate over whether there has been a real increase in atopy over the last decades, it is indisputable that there has been a dramatic change over the last 200 years. While isolated cases of anaphylaxis had been noted before, allergies were unheard of until the early 19th century. Only 54 years after the first description of hay fever in England in 1819, it was considered an epidemic and a clear association with urbanization had been reported.1 What aspect of urbanization is involved has been much debated. There has been much discussion amongst both experimental immunologists and epidemiologists over whether pollutants are responsible for increased allergic sensitization and/or severity.2,3 In the last few years new studies have supported an alternative explanation, namely the decline in infectious diseases in industrialized countries.4,5 Does this ‘hygiene theory’ toll the death knell for the ‘pollution theory’? In this review I will argue that there is a compelling case to be made for both theories and that even this is not the entire picture.

The effect of monocyte chemotactic protein 1 in intraperitoneal adhesion formation in a mouse model

OBJECTIVE: Intraperitoneal adhesions are a significant cause of morbidity among women of reproductive age. Monocyte chemotactic protein 1 plays a role in the chemotaxis of mononuclear cells and fibroblasts into the peritoneal injury site. To evaluate its role in intraperitoneal adhesion formation, we used an established postsurgical adhesion model in mice. STUDY DESIGN: Surgical adhesions in mice were induced by scraping and crushing peritoneal sites. We analyzed the injury sites for the temporal expression of monocyte chemotactic protein 1 messenger ribonucleic acid and cellular infiltration at 12 to 24 hours across 10 days and evaluated the effects of monocyte chemotactic protein 1 and anti–monocyte chemotactic protein 1 neutralizing antibody on adhesion formation. On induction of adhesions animals were randomly assigned to 1 of 4 groups: (1) control, (2) nonspecific immunoglobulin G, (3) monocyte chemotactic protein 1, (4) anti–monocyte chemotactic protein 1 antibody. Animals received daily intraperitoneal injections for 6 days. Adhesions were scored on day 14 and immuno-stained for fibroblasts and macrophages. RESULTS: Adhesions developed consistently by the fifth postoperative day. We detected an increase in monocyte chemotactic protein 1 messenger ribonucleic acid expression at 48 hours; this remained until the fourth postoperative day. By the second day macrophages were present at the injury site. Animals treated with anti–monocyte chemotactic protein 1 antibody had significantly fewer adhesions develop than did the other three groups. CONCLUSION: This study demonstrates that monocyte chemotactic protein 1 may play a role in adhesion formation. Inhibiting the action of this chemokine may help to prevent adhesions. (Am J Obstet Gynecol 1998;179:438-43.)

The role of monocyte chemotactic protein-1 in intraperitoneal adhesion formation.

Abdomino-pelvic adhesions arise from infection, endometriosis, or peritoneal injury during surgery, and represent a significant source of morbidity in women of reproductive age. Monocyte chemotactic protein-1 (MCP-1) plays a role in the chemotaxis of mononuclear cells and fibroblasts in a murine wound repair model. To evaluate the role of MCP-1 in intraperitoneal adhesion formation, we investigated peritoneal fluid MCP-1 levels of women undergoing laparoscopy. Patients without endometriosis were divided into two groups: normal fertile women undergoing bilateral tubal ligation without intraperitoneal adhesions (n=14) and women with pelvic adhesions (n=8). Patients with endometriosis were arranged into two groups: women with (n=17) and without (n=17) adhesions. Peritoneal fluid MCP-1 levels were quantified using an enzyme-linked immunosorbent assay (ELISA). Peritoneal biopsy samples were immunostained for the detection of MCP-1 protein and macrophages, and were also processed for the presence of MCP-1 mRNA expression. Among women without endometriosis, the median peritoneal fluid MCP-1 level was 144 pg/ml (range 54-261) in women without adhesions and was 336 pg/ml (range 130-2494) in women with adhesions (P=0.01). There was a significant correlation between adhesion scores and MCP-1 levels (r=0.50; P=0.018). Among women with endometriosis, peritoneal fluid MCP-1 levels significantly correlated with the stage of the disease. The presence or absence of adhesions did not significantly affect the peritoneal fluid MCP-1 levels in this group of women. In summary, we have found that women with adhesions have elevated peritoneal fluid MCP-1 levels. However, we were not able to show an incremental effect of adhesions on peritoneal fluid MCP-1 levels of patients with endometriosis. Thus, we conclude that factors besides the intraperitoneal adhesions contribute to the elevated peritoneal fluid MCP-1 levels in patients with endometriosis.

Role of alpha L beta 2 integrin avidity in transendothelial chemotaxis of mononuclear cells.

The leukocyte integrin alpha L beta 2 (LFA-1) is important in transendothelial migration. Since it is not fully understood how LFA-1 mediates transmigration, we studied the effects of alpha L and beta 2 cytoplasmic domain mutants that alter LFA-1 adhesiveness for intercellular adhesion molecule-1. Monocyte chemotactic protein-1 (MCP-1) induced LFA-1-dependent transendothelial migration of Jurkat and J-beta 2.7 transfectants coexpressing the MCP-1 receptor CCR2B and wild-type alpha L. No transendothelial chemotaxis was observed with truncation mutants of the alpha L cytoplasmic tail, which rendered LFA-1 constitutively active or locked LFA-1 in a low avidity state unresponsive to cellular activation. Moreover, transendothelial chemotaxis of lymphoblastoid SLA transfectants was abolished by truncation of the beta 2 cytoplasmic domain, but not by mutation of its TTT motif, which is important in phorbol ester-induced adhesion. These data indicate that transmigration may require both alpha L and beta 2 cytoplasmic domains. We further show that MCP-1-induced transendothelial chemotaxis of PBMC was inhibited by sustained activation of LFA-1 with Mn2+ or a stimulatory mAb to beta 2. Dimeric soluble intercellular adhesion molecule-1 also reduced transendothelial chemotaxis of PBMC. Taken together, our data suggest that transendothelial chemotaxis of mononuclear cells may involve dynamic changes in LFA-1 avidity.