Strain and model dependent differences in inflammatory cell recruitment in mice

OBJECTIVE AND DESIGN: The objective of this study was to determine genetic differences in inflammation in these distinct inbred mouse strains. METHODS: Peritoneal leukocyte recruitment, matrix metalloproteinases and cytokines were quantified in A/J, 129/svJ, C57BL/6J, using thioglycollate or biomaterial implants as inflammatory stimuli. RESULTS: In response to thioglycollate, A/J had significant decreases compared to C57BL/6J in both neutrophil (86 %) and macrophage (62 %) recruitment, and 129/svJ had a significant (43 %) decrease compared to C57BL/6J in macrophage recruitment. The reduced leukocyte recruitment corresponded to reduced matrix metalloproteinase-9. In the bioimplant model, 129/svJ had a 2-fold increase in neutrophil and macrophage recruitment compared to C57BL/6J, and the increased leukocyte recruitment corresponded to elevated cytokines, monocyte inhibitory protein-2 and monocyte chemoattractant protein-1, in the lavage compared to the values for C57BL/6J. CONCLUSION: Not only was leukocyte recruitment strain dependent, but the three strains had marked differences in metalloproteinases and cytokine response. In addition, there were model specific differences in the metalloproteinase and cytokine response to the two inflammatory stimuli. Thus, inflammatory cell recruitment is genetically determined and stimulus specific and may determine the susceptibility to complex diseases.     

Role of interleukin-18 in intrahepatic inflammatory cell recruitment in acute liver injury

Although the innate immune system has been demonstrated to play important roles as the first line of defense against various infections, little is known about the interactions between intrahepatic inflammatory cells and the cytokine network in the liver. Here, we examined the role of IL-18 in IHL recruitment in acute liver injury. C57BL/6 mice were injected with an αCD40 mAb, and their serum IL-18 levels were observed to increase, with subsequent recruitment of IHLs into the liver. NKT cells were involved in this liver injury, as the serum ALT levels were reduced in NKT KO mice through the suppression of macrophage and monocyte migration and cytokine production. In contrast, depletion of neutrophils exacerbated the liver injury associated with high levels of TNF-α and IL-18 and increased numbers of macrophages and monocytes. Treatment with a neutralizing antibody against IL-18 reduced the serum ALT levels and inflammatory cell accumulation in the liver. Finally, additional administration of rIL-18 with αCD40 injection caused severe liver injury with increased IFN-γ production by NK cells. In conclusion, these findings demonstrate that IL-18 modulates liver inflammation by the recruitment of inflammatory cells, including NKT cells, macrophages, monocytes, and neutrophils.     

Effects of cigarette smoke and alcohol on ciliated tracheal epithelium and inflammatory cell recruitment

Ciliated epithelium represents the first line of host defense against lung infection. Most alcoholics smoke and are at high risk for developing lung infections. We reported that cigarette smoke activates protein kinase C (PKC) and alcohol desensitizes ciliary beat frequency (CBF) to beta-agonists in bovine bronchial epithelial cells in vitro. The combined effect of smoke and alcohol exposure on mouse ciliated tracheal epithelium has not been studied in vivo. We hypothesized that previously observed in vitro effects of smoke and alcohol exposure could be replicated in vivo. Female C57BL/6 mice were exposed to whole body cigarette smoke only, 20% alcohol ad libitum in drinking water only, or the combination of cigarette smoke plus alcohol for 6 wk. Bronchoalveolar lavage (BAL) cell populations, CBF, and airway kinase activity were assessed. Total BAL cells were decreased in animals exposed to alcohol alone and increased in animals exposed to smoke alone. Mice receiving smoke and alcohol had cell levels similar to smoke alone. Baseline CBF was not affected in any group; however, isoproterenol stimulation of CBF was blunted by alcohol exposure and actually slowed below baseline in the smoke plus alcohol group. Isoproterenol-induced PKA activity was inhibited in mice receiving alcohol independent of smoke exposure. Smoke activated PKC independent of alcohol. The isoproterenol-induced slowing below baseline of CBF after combined smoke and alcohol exposure demonstrates a novel ciliary impairment likely related to the combination of alcohol-mediated PKA desensitization and smoke-stimulated PKC activation, possibly through acetaldehyde present in the vapor phase of cigarette smoke.     

Leukocyte recruitment and the acute inflammatory response

Leukocyte recruitment is a hallmark feature of the inflammatory response. This review summarizes the generally accepted paradigm of leukocyte recruitment based on studies using intravital microscopy to visualize the microcirculation. The role of selectins and alpha4-integrin in rolling as well as integrin-mediated adhesion is discussed. However, it is becoming increasingly clear that the recruitment cascade within organs differs and therefore the review also attempts to highlight what is and is not known regarding leukocyte recruitment into the brain microvasculature. In the second part of this review, we provide some discussion of mechanisms by which the inflammatory response may be terminated. Particular emphasis on nuclear factor Nf kappaB and how IL10, IL13 and secreted leukocyte protease inhibitor (SLPI) may impact upon the Nf kappaB-dependent inflammatory response is presented.     

CD28 and CTLA4 coordinately regulate airway inflammatory cell recruitment and T-helper cell differentiation after inhaled allergen

Airway inflammation after inhaled allergen exposure requires the recruitment, activation, and differentiation of antigen-specific T cells into T helper (Th) 2 effector cells. These processes are regulated not only by antigen engagement of the T-cell receptor, but also by specific accessory molecules on the surface of the T cell. We examined how the balance of signals derived through the CD28 and cytotoxic T-lymphocyte antigen (CTLA) 4 receptors modulate the outcome of inhaled antigen exposure in a murine model of allergic airway inflammation. Mice deficient in CD28 have defective Th2 cell development and failed to develop inflammation after sensitization and inhaled challenge with ovalbumin. Prevention of B7-CTLA4 interactions in CD28-deficient mice restored lymphocyte but not eosinophil recruitment to the airway. Analysis of cytokine gene expression revealed that T cells from CD28-deficient mice failed to differentiate into Th2 cells in either the presence or absence of B7-dependent signals, and therefore did not recruit eosinophils to the airway. Thus, the processes of T-cell recruitment to the airway and T-cell differentiation have distinct requirements for signals mediated through the CD28 and CTLA4 receptors, demonstrating that these receptors are important regulatory components in the development of allergic airway inflammation.     

Leukocyte recruitment and turnover in an experimental inflammatory exudate

The turn-over of leukocytes at sites of inflammation in vivo is to a large extent uninvestigated, mainly due to the technical difficulties associated with sampling and analysis of the inflammatory exudate. This paper investigates the immigration of fluorescently labeled granulocytes into exudate chambers at 8 h and at 1, 3, and 6 days after implantation into abdominal muscle of rat. In each experiment, the circulating granulocytes were labeled by intravenous administration of the DNA-labeling fluorochrome Hoechst 33342^® and allowed to migrate into the chamber during 6 h before harvesting the chamber exudate. The rate of granulocyte immigration into the chamber varied considerably over time, showing a minimum at 3 days after implantation. The resulting kinetic pattern of granulocyte numbers in the exudate showed a two-step appearance, different from that of earlier determinations in soft tissue. A comparison between the calculated rates of granulocyte immigration and the total number of granulocytes present in the exudate at different times indicated that all immigrated cells survived in the chamber for the entire observation period of 6 days.     

Galectins: versatile modulators of cell adhesion, cell proliferation, and cell death

 Lectins, or carbohydrate binding proteins, recognize specific oligosaccharide structures on glycoproteins and glycolipids. Several families of animal lectins have been identified; for some of these lectins, functions such as leukocyte adhesion and microbial opsonization have been described. The galectins are a family of lectins found in species ranging from sponges and nematodes to humans. Members of the galectin family have been proposed to mediate cell adhesion, to regulate cell growth, and to trigger or inhibit apoptosis. The expression pattern of different galectins changes during development, and this pattern is also altered at sites of inflammation and in breast, colon, prostate, and thyroid carcinomas. In addition, the level of expression of some galectins by tumor cells has been shown to be correlated with metastatic potential. The mechanisms by which galectins exert these diverse effects remain largely unknown. Some glycoprotein counterreceptors recognized by certain galectins have been identified; this is an important first step in understanding the cell-type specific effects of different galectins. This review discusses the way in which the modulation of galectin activity may affect strategies for treatment of a variety of human diseases, including autoimmunity and cancer. Received: 4 April 1997 / Accepted: 24 September 1997     

Ontogeny of inflammatory cell responsiveness

Newborn calves, like human infants, are uniquely susceptible to bacterial infections. Part of this increased susceptibility may be related to defects in newborn polymorphonuclear leukocyte (PMN) defensive functions. It remains unclear whether reported deficits in newborn PMN function represent maturational disorders or are manifestations of some form of perinatal suppression phenomenon. We therefore compared the ability of bovine newborn PMNs (less than 24 h old), newborn PMNs (7–10 days of age), fetal PMNs (210–220 days gestational age), and adult PMNs to generate superoxide anion (O ₂ ^– ) as an indicator of respiratory burst activity. Citrated biood was collected, and PMNs were isolated to greater than 95% purity and 98% viability. O ₂ ^– generation was measured as the superoxide dismutase-inhibitable (10 g/ml) reduction of ferricytochrome c (2 mg/ml) after activation of PMNs with phorbol myristate acetate (PMA, 2 g/ml) to directly stimulate protein kinase C. The reaction kinetics were measured (37°C, 550 nm) using a spectrophotometer and chart recorder for continuous monitoring. O ₂ ^– generation was measured for 5 min after the initial lag period and the total nanomoles of O ₂ ^– generated calculated using the extinction coefficient for ferricytochromec. Newborn PMNs (N=10) generated significantly less O ₂ ^– (5.7 ±0.8 nmol O ₂ ^– /10⁶ cells/5 min,P < 0.01) than did adult PMNs (N=14) (9.6 ±2.1 nmol O ₂ ^– /10¹⁰ cells/5 min) or fetal PMNs (N=4) (10.7 ±0.7 nmol O ₂ ^– /10⁶ cells/5 min). PMNs from 7-to 10-day-old calves (N=9) generated almost identical amounts of O ₂ ^– as newborn PMNs (5.7 ±1.6 nmol O ₂ ^– /10⁶ ceils/5 min). There was no difference in measured lag time period between new-born and adult PMNs, but fetal PMNs had significantly reduced (P < 0.01) mean lag time. The data indicated that bovine newborn PMNs have a decreased ability to generate O ₂ ^– in response to PMA stimulation, which persists for at least 7–10 days, and that this functional decrement may be a manifestation of some form of perinatal PMN suppression phenomenon rather than a developmental abnormality since fetal PMNs produced O ₂ ^– as well as adult PMNs.     

Macrophage inflammatory protein-1alpha relates to the recruitment of inflammatory cells in myosin-induced autoimmune myocarditis in rats.

In experimental autoimmune myocarditis (EAM) there is a characteristic initial focal inflammatory response in the myocardium, induced mainly by CD4(+) T cells and macrophages, which leads to massive myocardial damage. Macrophage inflammatory protein-1alpha (MIP-1alpha) induces chemotaxis in lymphocytes, eosinophils, basophils, and macrophages. We assessed the potential role of MIP-1alpha in the pathogenesis of EAM in rats immunized with porcine myosin. Following immunization, the levels of MIP-1alpha mRNA in EAM showed an increase on Day 11 and peaked on Day 17. MIP-1alpha-positive cells were predominantly immunoreactive to OX6 antibody (dendritic cells) and ED2 antibody (resident macrophages) by Day 14. Marked cellular infiltration was seen on Day 17 with the major population of MIP-1alpha-positive cells also positive for ED1 (inflammatory macrophages). We then examined the association of MIP-1alpha with the development of myocardial inflammation. Rats were divided into three groups: Group A consisted of EAM rats (n = 10); Group B consisted of EAM rats treated with anti-MIP-1alpha (1 mg/kg) on Days 11, 13, and 15, before the onset of initial inflammation (n = 5); and Group C consisted of EAM rats treated with anti-MIP-1alpha from the start of the initial inflammation on Days 14, 16, and 18 (n = 5). Rats were euthanized on Day 21 and three transverse sections of the heart were prepared to determine the percentage of the area affected by inflammatory lesions. This area of inflammation was significantly smaller in Group B (27 +/- 4%) than in Groups A (51 +/- 6%) or C (50 +/- 6%) (p < 0.01), indicating that the administration of antibody before the initiation of inflammation, in part, will inhibit myocardial inflammation. These data suggest that MIP-1alpha may play an important role in the recruitment of inflammatory cells in the early stages of EAM.     

Endogenous ligands of Toll-like receptors: implications for regulating inflammatory and immune responses

Toll-like receptors (TLRs) have a crucial role in regulating immunity against microbial agents. Recent studies indicate that these receptors might also have an important role in regulating responses to endogenous stimuli, such as necrotic cells, heat-shock proteins and extracellular matrix breakdown products. Specifically, TLR2 and TLR4 were shown to mediate expression of inflammatory genes and trigger dendritic-cell 'maturation' by these agents. These intriguing findings suggest that the ancient family of TLRs are involved in the recognition, not only of microbes, but also of endogenous harmful stimuli. However, potential complications associated with microbial contamination of endogenous agents and the specific nature of in vivo responses induced by these agents remain to be determined.     

Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin

Stressed cells undergoing necrosis release molecules that acts as endogenous danger signals to alert and activate innate immune cells. Both HMGB1 and HSP70 are induced in activated monocytes/macrophages and also are released from stressed or injured cells. We investigated whether HMGB1 and HSP70 released from necrotic monocytes/macrophages, can act as danger signals to mediate proinflammatory cytokine responses to bacterial endotoxin or lipopolysaccharide (LPS). We show that cell lysate, obtained from necrotic cells directly stimulates the proinflammatory cytokine and chemokine responses in human monocyte/macrophage cell line, THP-1, as revealed by the induction of TNF-alpha, IL-6 and IL-8 mRNA expression and protein production. In the presence of LPS, necrotic cell lysate induced a more robust increase in all three proteins. We found that HMGB1 and HSP70 were indeed present in the necrotic cell lysate and were responsible for the significant induction of the proinflammatory cytokine expression, as neutralization with antibodies against both proteins blocked the increase in the cytokine production seen after incubating LPS-stimulated cells with the necrotic cell lysate. We also found that the newly identified triggering receptor expressed on myeloid cells-1 (TREM-1) was involved in mediating the HMGB1- and HSP70-induced cytokine production. Blocking TREM-1 on THP-1 cells with a recombinant chimera prevented the increase in cytokine production, while simultaneous blocking of TLR4 and TREM-1 completely abolished the proinflammatory response, suggesting that TREM-1 synergizes with TLR4 to mediate the effects of such signals from necrotic cells. In addition, blocking HMGB1 or HSP70 simultaneously with TREM-1 did not decrease the cytokine level further, confirming the involvement of TREM-1 in mediating the effect of HMGB1 and HSP70. Although the interaction of HMGB1 and HSP70 with TREM-1 induced I kappa B alpha and p38 expression, both of which are required for the inflammatory cytokine expression, blockade of TREM-1 did not affect I kappa B alpha expression but markedly reduced p38 activation, as revealed by Western blot analysis. Together, these results demonstrate that HMGB1 and HSP70 released from necrotic cells function as endogenous danger signals to augment the proinflammatory responses in monocytes/macrophage and that TREM-1 relays such signals to the cytokine expression cascade. This mechanism may contribute to the amplification and persistence of the inflammatory response to bacterial infection.     

Memory T cell recruitment to the lung airways

Memory T cells play an important role in the resolution of secondary respiratory virus infections through the production of anti-viral cytokines and the lysis of infected cells. Within the lung airways, memory T cells form an initial line of defense against secondary virus challenge by limiting early viral replication at the site of infection. Recent studies have addressed the generation and maintenance of airway memory T cells and their relationship with memory T cells in the secondary lymphoid organs. These advances have demonstrated that different memory subsets contribute to the maintenance of the airway memory T cell population and have identified molecules that influence trafficking to the lung tissue and the lung airways.     

Role of extracellular matrix proteoglycans in immune cell recruitment

Leucocyte recruitment is a critical component of the immune response and is central to our ability to fight infection. Paradoxically, leucocyte recruitment is also a central component of inflammatory‐based diseases such as rheumatoid arthritis, atherosclerosis and cancer. The role of the extracellular matrix, in particular proteoglycans, in this process has been largely overlooked. Proteoglycans consist of protein cores with glycosaminoglycan sugar side chains attached. Proteoglycans have been shown to bind and regulate the function of a number of proteins, for example chemokines, and also play a key structural role in the local tissue environment/niche. Whilst they have been implicated in leucocyte recruitment and inflammatory disease, their mechanistic function has yet to be fully understood, precluding therapeutic targeting. This review summarizes what is currently known about the role of proteoglycans in the different stages of leucocyte recruitment and proposes a number of areas where more research is needed. A better understanding of the mechanistic role of proteoglycans during inflammatory disease will inform the development of next‐generation therapeutics.     

Physiological contribution of CD44 as a ligand for E-Selectin during inflammatory T-cell recruitment

Endothelial selectins guide the migration of inflammatory T cells to extralymphoid tissues. Whereas P-selectin glycoprotein ligand-1 (PSGL-1) functions as the exclusive ligand for P-selectin, it acts in coordination with additional glycoproteins to mediate E-selectin binding. CD44 can act as one such ligand in neutrophils, but its contribution in inflammatory T lymphocytes remains unexplored. We have used real-time in vivo imaging of the cremasteric and dermal microcirculations to explore the dynamics of leukocyte recruitment, as well as the physiological contribution of CD44 in a model of Th1-driven inflammation. CD4(+) T-cell rolling frequency and kinetics, as well as arrest, were dependent on endothelial selectins and were markedly altered under inflammatory conditions. CD44 extracted from Th1 cells bound to soluble E-selectin in vitro and cooperated with PSGL-1 by controlling rolling velocities and promoting firm arrest. Using several competitive recruitment assays in a delayed-type hypersensitivity model, we show that the combined absence of CD44 and PSGL-1 impairs inflammatory T-cell recruitment beyond that of PSGL-1 alone. Differential expression of leukocyte fucosyltransferases in these cells may account for the differential use of E-selectin ligands relative to neutrophils. Our results identify additional mechanisms by which CD44 modulates the inflammatory response.     

Adaptor proteins: Flexible and dynamic modulators of immune cell signalling

To maintain homeostasis, all cells respond to environmental cues via a multitude of surface receptors. In order to act appropriately in their environment, cells are dependent on the transduction of the incoming signal through tightly regulated and interconnected signalling pathways to the cell nucleus. In particular, cells implicated in the immune system greatly depend on such systems to respond in a flexible and dynamic manner to environmental challenges. One major group of intracellular proteins that are involved in these signalling pathways are adaptor proteins. Although adaptor proteins are essential for normal immune cell operation, the functional role of this group of signalling proteins remains to be fully appreciated. So far, research on adaptor proteins has revealed their unique potential in building transient complexes in a reversible, dynamic and inducible manner. In this review, we explore the roles of adaptor proteins – in space and time of intracellular signalling – and their associations with human disease. Examples of adaptor proteins expressed in hematopoietic cells highlight their crucial role in the immune system. Lastly, we present challenges faced in elucidating roles of adaptor proteins, as illustrated by the T cell–specific adaptor (TSAd) protein encoded by the SH2D2A gene.