Blockade of PD-1/PD-L1 Pathway Enhances the Antigen-Presenting Capacity of Fibrocytes.

Fibrocytes, a distinct population of collagen-producing, monocyte-derived cells, are involved in wound healing as well as fibrotic diseases. Recently, fibrocytes have been revealed to play a role in the tumor microenvironment, particularly under antiangiogenic therapy. In addition, combination cancer immunotherapy with immune checkpoint inhibitor and antiangiogenic agents have been developed for various cancers in the clinical setting, although the immunological background is not clear. In the current study, we aimed to determine the function of fibrocytes in tumor immunity induced by immune checkpoint inhibitor therapy. Human and murine fibrocytes were generated from PBMCs and lungs, respectively. The expression of costimulatory and inhibitory molecules on fibrocytes was examined by flow cytometry. The stimulation of CD8+ T cells by fibrocytes was examined in MLRs with a 3H-thymidine incorporation assay. Fibrocytes expressed CD80low and CD86high as a costimulatory molecule, and expressed PD-L1high, but not PD-L2, as a coinhibitory molecule. Without any stimulation, fibrocytes strongly enhanced the proliferation of CD8+ T cells in mice and humans. Treatment with anti-CD86 and -CD54 Abs inhibited the growth of CD8+ T cells induced by fibrocytes. Anti-PD-L1 Ab further enhanced the proliferation of CD8+ T cells, even in the OVA-specific MLR with OT-1Rag-/- mice. Importantly, fibrocytes derived from PBMCs of patients with lung adenocarcinoma or murine MC38 tumors augmented the proliferation of CD8+ T cells with PD-L1 blockade. These results suggest that fibrocytes infiltrating tumor sites may play a role in the antitumor immunity mediated by CD8+ T cells when the activity is further enhanced by PD-L1/PD-1 blockade.

Characterization of epithelial cells, connective tissue cells and immune cells in human upper airway mucosa by immunofluorescence multichannel image cytometry: a pilot study.

Epithelial, connective tissue and immune cells contribute in various ways to the pathophysiology of chronic rhinosinusitis (CRS). However, data of their distribution in upper airway mucosa are sparse. We aimed to provide quantitative, purely informative data on the distribution of these cell lineages and their coexpression patterns, which might help identifying, e.g., cells in the epithelium undergoing through epithelial-mesenchymal transition (EMT). For this purpose, we used immunofluorescence multichannel image cytometry (IMIC). We examined fixed paraffin-embedded tissue samples (FFPE) of six patients with chronic rhinosinusitis (CRS) and of three patients without CRS (controls). The direct-conjugated antibodies pancytokeratin, vimentin and CD45/CD18 were used for coexpression analysis in epithelial layer and lamina propria. Image acquisition and analysis were performed with TissueFAXS and StrataQuest, respectively. To distinguish positive from negative expression, a ratio between cell-specific immunostaining intensity and background was developed. Isotype controls were used as negative controls. Per patient, a 4.5-mm2 tissue area was scanned and a median of 14,875 cells was recognized. The most common cell types were cytokeratin-single-positive (26%), vimentin-single-positive (13%) and CD45/CD18-single-positive with CD45/CD18-vimentin-double-positive cells (29%). In the patients with CRS, CD45/CD18-single-positive cells were 3-6 times higher compared to the control patients. In the epithelial layer, cytokeratin-vimentin-double-positive EMT cells were observed 3-5 times higher in the patients with CRS than in the control patients. This study provided quantitative data for the distribution of crucial cell types in CRS. Future studies may focus on the distribution and coexpression patterns of different immune cells in CRS or even cancer tissue.

Pathogenic Roles of Autoantibodies and Aberrant Epigenetic Regulation of Immune and Connective Tissue Cells in the Tissue Fibrosis of Patients with Systemic Sclerosis.

Systemic sclerosis (SSc) is a multi-system autoimmune disease with tissue fibrosis prominent in the skin and lung. In this review, we briefly describe the autoimmune features (mainly autoantibody production and cytokine profiles) and the potential pathogenic contributors including genetic/epigenetic predisposition, and environmental factors. We look in detail at the cellular and molecular bases underlying tissue-fibrosis which include trans-differentiation of fibroblasts (FBs) to myofibroblasts (MFBs). We also state comprehensively the pro-inflammatory and pro-fibrotic cytokines relevant to MFB trans-differentiation, vasculopathy-associated autoantibodies, and fibrosis-regulating microRNAs in SSc. It is conceivable that tissue fibrosis is mainly mediated by an excessive production of TGF-ß, the master regulator, from the skewed Th2 cells, macrophages, fibroblasts, myofibroblasts, and keratinocytes. After binding with TGF-ß receptors on MFB, the downstream Wnt/ß-catenin triggers canonical Smad 2/3 and non-canonical Smad 4 signaling pathways to transcribe collagen genes. Subsequently, excessive collagen fiber synthesis and accumulation as well as tissue fibrosis ensue. In the later part of this review, we discuss limited data relevant to the role of long non-coding RNAs (lncRNAs) in tissue-fibrosis in SSc. It is expected that these lncRNAs may become the useful biomarkers and therapeutic targets for SSc in the future. The prospective investigations in the development of novel epigenetic modifiers are also suggested.

CD4 T-cell hyporesponsiveness induced by schistosome larvae is not dependent upon eosinophils but may involve connective tissue mast cells.

In areas endemic for schistosomiasis, people can often be in contact with contaminated water resulting in repeated exposures to infective Schistosoma mansoni cercariae. Using a murine model, repeated infections result in IL-10-dependent CD4(+) T-cell hyporesponsiveness in the skin-draining lymph nodes (sdLN), which could be caused by an abundance of eosinophils and connective tissue mast cells at the skin infection site. Here, we show that whilst the absence of eosinophils did not have a significant effect on cytokine production, MHC-II(+) cells were more numerous in the dermal cell exudate population. Nevertheless, the absence of dermal eosinophils did not lead to an increase in the responsiveness of CD4(+) T cells in the sdLN, revealing that eosinophils in repeatedly exposed skin did not impact on the development of CD4(+) T-cell hyporesponsiveness. On the other hand, the absence of connective tissue mast cells led to a reduction in dermal IL-10 and to an increase in the number of MHC-II(+) cells infiltrating the skin. There was also a small but significant alleviation of hyporesponsiveness in the sdLN, suggesting that mast cells may have a role in regulating immune responses after repeated exposures of the skin to S. mansoni cercariae.

Elevations in T-helper-2-initiating cytokines (interleukin-33, interleukin-25 and thymic stromal lymphopoietin) in lesional skin from chronic spontaneous ('idiopathic') urticaria.

BACKGROUND: The mechanism of wealing in chronic spontaneous urticaria (CSU) is largely unknown. We previously demonstrated increased expression of T-helper 2 [interleukin (IL)-4 and IL-5] cytokines in skin biopsies from CSU. This suggested that Th2-initiating cytokines [IL-33, IL-25 and thymic stromal lymphopoietin (TSLP)], released through innate immune mechanisms, may play a role in pathogenesis. OBJECTIVES: To identify Th2-initiating cytokines in lesional and nonlesional skin from patients with CSU and to compare the results with a control group. METHODS: Paired biopsies (one from a 4-8 h spontaneous weal and one from uninvolved skin) were taken from eight patients with CSU and nine control subjects, and studied by immunohistochemistry and confocal microscopy. RESULTS: There were increases in IL-4(+) and IL-5(+) cells in lesional skin vs. controls (P = 0·03 and P < 0·001, respectively) and marked elevations in the numbers of IL-33(+), IL-25(+) and TSLP(+) cells in the dermis of lesional skin vs. both nonlesional skin (P = 0·002, P = 0·01 and P = 0·04, respectively) and controls (P = 0·001, P < 0·001 and P = 0·005, respectively). There was also a correlation between the numbers of IL-33(+) and IL-25(+) cells (r = 0·808, P = 0·015). IL-33 localized to CD31(+) endothelial cells, CD90(+) fibroblasts, CD68(+) macrophages and tryptase(+) mast cells, whereas IL-25 was expressed by epithelial cells, mast cells and major basic protein-positive eosinophils. IL-33 and IL-25 were constitutively expressed in the epidermis of both controls and patients with CSU. CONCLUSIONS: Increased expression of Th2-initiating cytokines in lesional skin in CSU suggests that innate pathways might play a role in the mechanism of wealing. As Th2-initiating cytokines play a role in mast cell activation, inflammation and vascular leakage in CSU, these findings may also have therapeutic implications.

The role of Toll-like receptor 2 and 4 in gingival tissues of chronic periodontitis subjects with type 2 diabetes.

BACKGROUND AND OBJECTIVE: Diabetes is one important risk factor of chronic periodontitis. However, the roles of toll-like receptor (TLR) 2 and TLR4, which are implicated in the inflammatory process in both chronic periodontitis and diabetes, have not been studied. This study aimed to determine whether TLR2 and TLR4 might be involved in the relationship between chronic periodontitis and diabetes by examining TLR2 and TLR4 expression in gingival tissues from subjects with chronic periodontitis without diabetes (CP) and with diabetes (CP+DM) and from periodontally healthy subjects without diabetes (PH) and with diabetes (PH+DM). MATERIAL AND METHODS: Gingival tissues were collected from 23 CP subjects, 21 CP+DM subjects, 22 PH subjects and 20 PH+DM subjects. The expression of TLR2 and TLR4 in gingival tissues was determined using an immunohistochemical method. In gingival epithelium, staining patterns and intensity levels of TLR2 and TLR4 expression were studied. In connective tissues, the percentages of TLR2- and TLR4-positive cells were calculated. The intensity levels and the percentages of positive cells were statistically analyzed. RESULTS: Chronic periodontitis or diabetes showed no significant effect on TLR2 expression in the oral epithelium. However, diabetes increased the expression of TLR2 in sulcular epithelium and changed the pattern of TLR2 expression in gingival epithelium. Chronic periodontitis decreased the expression of TLR4 in gingival epithelium. In connective tissue under sulcular epithelium, CP+DM subjects showed statistically significant higher percentages of TLR2- and TLR4-positive cells compared with PH and PH+DM subjects. CONCLUSION: Our results suggest that hyperglycemia and chronic periodontitis had effects on TLR2 and TLR4 expression in gingival tissue. The differences in TLR2 and TLR4 expression could contribute to a greater inflammatory response, leading to periodontal disease initiation and progression.

GATA-1 as a regulator of mast cell differentiation revealed by the phenotype of the GATA-1low mouse mutant.

Here it is shown that the phenotype of adult mice lacking the first enhancer (DNA hypersensitive site I) and the distal promoter of the GATA-1 gene (neo Delta HS or GATA-1(low) mutants) reveals defects in mast cell development. These include the presence of morphologically abnormal alcian blue(+) mast cells and apoptotic metachromatic(-) mast cell precursors in connective tissues and peritoneal lavage and numerous (60-70% of all the progenitors) "unique" trilineage cells committed to erythroid, megakaryocytic, and mast pathways in the bone marrow and spleen. These abnormalities, which were mirrored by impaired mast differentiation in vitro, were reversed by retroviral-mediated expression of GATA-1 cDNA. These data indicate an essential role for GATA-1 in mast cell differentiation.

Fibrocyte activation in rheumatoid arthritis.

OBJECTIVES: RA is a common, relapsing autoimmune disease primarily affecting the joints. Fibroblast-like synovial (FLS) cells are thought to be responsible for pannus formation and secretion of factors that recruit leucocytes to affected joints, thereby promoting bone and cartilage destruction. Fibrocytes are multipotent circulating stem cells that may have a role in RA pathogenesis, perhaps as the precursors of the FLS cells, or by regulating FLS cell function. METHODS: We utilized multidimensional phospho-specific flow cytometry to characterize the activation status of peripheral blood (PB) fibrocytes derived from human RA patients at different stages of disease and from mice with CIA. RESULTS: Human PB fibrocytes from RA patients exhibited phosporylation activation of the p44/42 and p38 MAP kinases (MAPKs), and STAT3 (signal transducer and activator of transcription) and STAT-5 early in disease, within the first year of diagnosis. Similarly, in murine CIA, an increase in the total number of PB phosphoSTAT5-positive fibrocytes was observed at early time points in disease. Notably, in the affected paws of mice with CIA, we identified an increased number of fibrocytes, in contrast to the paws of control mice. CONCLUSIONS: These data suggest that activated fibrocytes may influence the disease process in RA and may serve as surrogate markers for disease in the PB of affected patients.

Brief Report: Group 3 Innate Lymphoid Cells in Human Enthesis.

OBJECTIVE: Group 3 innate lymphoid cells (ILC3s) play a pivotal role in barrier tissues such as the gut and the skin, two important sites of disease in spondyloarthritis (SpA). This study was undertaken to investigate whether normal or injured human enthesis, a key target tissue in early SpA, harbors ILC3s in entheseal soft tissue and adjacent perientheseal bone. METHODS: Interspinous ligament and spinous process bone from donors with no systemic inflammatory disease were collected, enzymatically digested, and immunophenotyped. The immunologic profile of entheseal cells was examined, and the transcriptional profile of sorted ILC3s was compared to that of ILC3s isolated from SpA synovial fluid (SF). To assess the ability of entheseal tissue to produce interleukin-17 (IL-17) and IL-22, entheseal digests were stimulated with IL-23 and IL-1ß. Osteoarthritic and ruptured Achilles tendon tissue was examined histologically. RESULTS: The proportion of ILCs in human entheseal soft tissue was higher than that in peripheral blood (P = 0.008); entheseal soft tissue and perientheseal bone both had a higher proportion of NKp44+ ILC3s (P = 0.001 and P = 0.043, respectively). Studies of retinoic acid receptor-related orphan nuclear receptor γt (RORγt), STAT3, and IL-23 receptor transcript expression validated the entheseal ILC3 phenotype. Cytokine transcript expression was similar in ILC3s isolated from enthesis and from SpA SF. Stimulation of normal entheseal digests with IL-23/IL-1ß led to up-regulation of IL-17A transcript, and histologic examination of injured/damaged entheses revealed the presence of RORγt-expressing cells. CONCLUSION: This work shows that human enthesis harbors a resident population of ILC3s, with the potential to participate in the pathogenesis of SpA.

Assessment of immunologic, proangiogenic and neurogenic properties of human peripheral nerve epineurium for potential clinical application.

The epineural sheath is a promising naturally occurring material for enhancement of peripheral nerve regeneration. Based on a literature search there is a limited number of reports on the biological and immunological properties of human epineurium. The goal of this study was to assess, using immunocytochemical methods, the immunological (HLA class I and II antigens, T lymphocytes, macrophages), proangiogenic (VEGF, CD31), and neurogenic (GFAP, S-100) properties of human epineurium isolated from ilioinguinal nerves (n=19) taken from deceased donors, and from sciatic nerves (n=12) taken from limbs amputated due to critical ischemia. Our studies confirmed reduced expression of HLA class II antigens on the infiltrating cells, a reduced number of T lymphocytes, and greater vessel density in the epineurium obtained from deceased organ donors. Macrophages were more abundant in the epineurium isolated from the amputated limbs. We found that the epineurium harvested from peripheral nerves of the deceased donors showed negligible immunogenic and increased proangiogenic properties compared to the epineurium of nerves taken from amputated limbs. These findings support the rationale to use human epineurium obtained from deceased donors as a new biological material for enhancement of peripheral nerve repair for potential clinical application in regenerative medicine.

Inflammation and cytokeratin 7/20 staining of cardiac mucosa in young patients with and without Helicobacter pylori infection.

BACKGROUND: Both Helicobacter pylori and gastro-oesophageal reflux disease (GORD) may cause inflammation in cardiac mucosa. Intestinal metaplasia (IM) is found more often in GORD associated inflammation than in inflammation caused by H pylori, especially in young individuals. AIM: To examine morphological differences in chronic inflammation in these two conditions by immunohistochemistry. PATIENTS/METHODS: Tissue blocks from cardiac mucosa of patients <45 years were available as follows: 10 patients with chronic inflammation of cardiac mucosa (carditis) and H pylori gastritis (group 1); 10 patients with (possibly GORD related) carditis, but normal antrum and corpus (group 2); and 10 patients with non-inflamed cardiac mucosa and normal antrum and corpus (group 3). Haematoxylin and eosin staining and immunohistochemical staining for various inflammatory cells were performed for patients in groups 1 and 2 as follows: CD20 (B cells), CD3 (T cells), CD4 (T helper cells), CD8 (T suppressor cells), CD163 (macrophages), CD138 (plasma cells), and CD117 (mast cells). For all patients, cytokeratin 7/20 (CK7/20) staining was performed. RESULTS: No clear differences were seen in the morphology of chronic inflammation between groups 1 and 2. In both, plasma cells were most abundant. CK7/20 staining showed no differences between these groups. CONCLUSION: Helicobacter pylori negative (possibly GORD associated) and H pylori related carditis cannot be distinguished on a morphological basis. The stronger tendency towards IM in the first entity cannot be explained by differences in the type of inflammation. Barrett-type CK7/20 staining seems typical for cardiac mucosa, irrespective of the type of inflammation or presence of IM.

Selective antibody blockade of lymphocyte migration to mucosal sites and mast cell adhesion.

The integrins alpha4beta7 and alpha4beta1 mediate adhesion to the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and the vascular cell adhesion molecule-1 (VCAM-1) and are important in T cell and allergic inflammatory reactions in the rat. The relative contributions of alpha4beta7 and alpha4beta1 in these reactions is unknown. To examine the role of alpha4beta7 in the rat a new mAb, TA-6, was developed. TA-6 inhibited adhesion to MAdCAM-1 but not to VCAM-1, a characteristic of alpha4beta7 adhesion, and immunofluorescence and immunoprecipitation studies were compatible with binding to alpha4beta7. TA-6 blocked rat lymphocyte adhesion to mesenteric lymph nodes and T cell migration to mucosal lymphoid tissues and it bound to rat mucosal mast cells. TA-6 did not inhibit lymphocyte adhesion to peripheral lymph nodes and T cell migration to peripheral lymphoid tissues or cutaneous inflammatory sites, and was not expressed on connective tissue mast cells.

Schistosoma mansoni egg-induced hepatic granulomas in mice deficient for the interferon-gamma receptor have altered populations of macrophages, lymphocytes and connective tissue cells.

Systemic production and mobilization of inflammatory cells and formation of hepatic periovular granulomas were studied in Schistosoma mansoni-infected mice with deficient interferon gamma (IFN-gamma) receptor (IFN-gammaR(o/o)). The impaired IFN-gamma signaling did not cause a significant modification of the overall kinetics of inflammatory cells, but mutant mice developed smaller hepatic periovular granulomas with a two-fold reduction in all the cell lineages. In granulomas of normal mice, the fully differentiated macrophages were progressively predominant, whilst in IFN-gammaR(o/o) mice, the granulomas contained a higher percentage of immature and proliferating monocytes. Granulomas of IFN-gammaR(o/o) mice had an enhanced and accelerated fibrotic reaction, corresponding to an increased content of proliferative and activated connective tissue cells. Simultaneously, their granulomas had an increased ratio of T over B cells, with an increase in CD8(+) and a reduction in CD4(+) T cells. The functional IFN-gamma receptor was not required for initial recruitment of monocytes and lymphocytes into granulomas, but it was necessary for the maturation of macrophages, upregulation of major histocompatibility class 2 (MHC-II) expression and consequent stimulation of lymphocyte subpopulations depending upon the MHC-II-mediated antigen presentation.

Quantitative analysis of immunocompetent cells in oral submucous fibrosis in Taiwan.

Previous studies have shown that the local and systemic upregulation of inflammatory and fibrogenic cytokines and downregulation of antifibrotic cytokines are central to the pathogenesis of oral submucous fibrosis (OSF). The immunocompetent cells, especially the macrophages and lymphocytes, are likely the main source of cytokine synthesis. Therefore, this study used an immunohistochemical method to quantify the T lymphocyte, B lymphocyte and macrophage densities in the epithelium and subepithelial connective tissue of 50 specimens of moderately advanced and advanced OSF and 10 specimens of normal oral mucosa (NOM). The mean T lymphocyte, B lymphocyte and macrophage densities in OSF specimens were 555.2+/-417.4, 63.4+/-44.3 and 66.9+/-76.4 cells/mm(2) in the subepithelial connective tissue and 308.1+/-261.1, 1.4+/-3.5 and 6.6+/-11.9 cells/mm(2) in the epithelium, respectively. These findings suggest that T lymphocytes were the major immunocompetent cells in both the subepithelial connective tissue and epithelium of OSF specimens. Macrophages and B lymphocytes are the minor immunocompetent cells in the subepithelial connective tissue and are only occasionally found in the epithelium of OSF specimens. Similar distribution of immunocompetent cells was also found in NOM specimens. However, the mean T lymphocyte, B lymphocyte and macrophage densities in the subepithelial connective tissue (271.2+/-107.0, 13.3+/-18.4 and 17.3+/-19.1 cells/mm(2), respectively) and the mean T lymphocyte density in the epithelium (97.7+/-51.4) of NOM specimens were significantly lower than the corresponding mean cell densities in OSF specimens. Using frozen tissue sections, we further quantified the CD4+ and CD8+ lymphocyte numbers in eight moderately advanced or advanced OSF specimens. It was found that the CD4+ and CD8+ lymphocyte densities were 213.3+/-140.7 and 101.5+/-72.8 cells/mm(2) in the subepithelial connective tissue of OSF specimens, respectively. The CD4+ to CD8+ lymphocyte ratio was 2.1:1. Our results showed a significant increase in the number of T lymphocytes and macrophages and a predominance of CD4+ lymphocytes over CD8+ lymphocytes in the subepithelial connective tissue of OSF specimens. We conclude that the cellular immune response may play an important role in the pathogenesis of OSF.

Development of different mast cell types in the opossum Didelphis albiventris.

Previous studies have disclosed three types of mast cell in opossums: connective tissue (CTMC), mucosal (MMC), and lymphatic sinus (LSMC). In contrast to most opossum lymph nodes, the mesenteric lymph node is virtually devoid of LSMC, displaying medullary cord CTMC. The present study aimed to describe the development of these mast cell populations. Toluidine blue staining and a histochemical method for demonstrating heparin allowed the identification of immature and mature mast cells. Immature CTMC devoid of detectable heparin were rare until postnatal day 10. Mature CTMC filled with heparin-containing granules became numerous by day 30 to day 40. In the ileum, despite the presence of mature CTMC in the submucosa and mucosa (villus base), immature mast cells first appeared in the villus core by day 65 and adult features were apparent by day 100. In LSMC-containing lymph nodes, immature mast cells were found in lymphatic sinuses by day 10. Clear signs of LSMC differentiation were observed from day 20. Compared with the 10-day value, the mean diameter of cytoplasmic granules at day 40 had doubled and that at day 110 had tripled. In the mesenteric lymph nodes, immature mast cells differentiated into lymphatic sinus CTMC-like cells. After day 80, most of them were located in medullary cords. Weaning and complete maturation of mucosa preceded the differentiation of MMC. In lymph nodes, LSMC differentiation occurred in parallel with the development of the medullary region and deep cortex units.

Lamina propria dendritic cells express activation markers and contact lymphocytes in chronic periodontitis.

BACKGROUND: Dendritic cells are characterized by shape, structure, and membrane molecule expression; they contact T lymphocytes to present antigens and stimulate plasma cell differentiation in vitro. Dendritic cells are known to be present in healthy human gingiva and to be altered in HIV-associated periodontitis. Here, we address the phenotype, location, and intercellular relationships of dendritic cells in chronic periodontitis. METHODS: Biopsies from patients with chronic periodontitis were analyzed by electron microscopy and indirect immunofluorescence for dendritic cells and lymphocyte markers. RESULTS: Langerhans' cells were spread in oral epithelium but restricted to the basal layer in pocket epithelium; they did not usually express major histocompatibility complex (MHC)-II antigens nor contact lymphocytes. Dendritic cells were abundant in the lamina propria of pocket epithelium; they were MHC-II positive, admixed with CD4-positive and CD8-positive T lymphocytes, and, they expressed CD54, CD80, and CD86. Dendritic cells often contacted lymphocytes and were also located within plasma cell aggregates. CONCLUSIONS: The data suggest that prerequisites for mounting a T cell-mediated immune response exist in chronic periodontitis, although this response is limited to the lamina propria. These results suggest that T-cell responses offer limited protection and can contribute to tissue damage during periodontal disease.

Multifunctional roles of reticular fibroblastic cells: more than meets the eye?

Fibroblastic reticular cells (FRCs) are stromal cells found in secondary lymphoid organ. Despite its structural function in the lymph nodes being well established, recent studies indicate that the FRCs also play a key role in immunological processes, associated with cell transit, immune response, and cells activation quality, and contribute to peripheral tolerance. To this end, we focus this review on lymph nodes FRC characterization and discuss functional aspects such as production of cytokines and chemokines and their involvement in the immune response, seeking to establish whether certain subsets have a more functional specialization.

Inflammation induces irreversible biophysical changes in isolated nucleus pulposus cells.

Intervertebral disc degeneration is accompanied by elevated levels of inflammatory cytokines that have been implicated in disease etiology and matrix degradation. While the effects of inflammatory stimulation on disc cell metabolism have been well-studied, their effects on cell biophysical properties have not been investigated. The hypothesis of this study is that inflammatory stimulation alters the biomechanical properties of isolated disc cells and volume responses to step osmotic loading. Cells from the nucleus pulposus (NP) of bovine discs were isolated and treated with either lipopolysaccharide (LPS), an inflammatory ligand, or with the recombinant cytokine TNF-α for 24 hours. We measured cellular volume regulation responses to osmotic loading either immediately after stimulation or after a 1 week recovery period from the inflammatory stimuli. Cells from each group were tested under step osmotic loading and the transient volume-response was captured via time-lapse microscopy. Volume-responses were analyzed using mixture theory framework to investigate two biomechanical properties of the cell, the intracellular water content and the hydraulic permeability. Intracellular water content did not vary between treatment groups, but hydraulic permeability increased significantly with inflammatory treatment. In the 1 week recovery group, hydraulic permeability remained elevated relative to the untreated recovery control. Cell radius was also significantly increased both after 24 hours of treatment and after 1 week recovery. A significant linear correlation was observed between hydraulic permeability and cell radius in untreated cells at 24 hours and at 1-week recovery, though not in the inflammatory stimulated groups at either time point. This loss of correlation between cell size and hydraulic permeability suggests that regulation of volume change is disrupted irreversibly due to inflammatory stimulation. Inflammatory treated cells exhibited altered F-actin cytoskeleton expression relative to untreated cells. We also found a significant decrease in the expression of aquaporin-1, the predominant water channel in disc NP cells, with inflammatory stimulation. To our knowledge, this is the first study providing evidence that inflammatory stimulation directly alters the mechanobiology of NP cells. The cellular biophysical changes observed in this study are coincident with documented changes in the extracellular matrix induced by inflammation, and may be important in disease etiology.

Effect of smoking on neutrophil apoptosis in chronic periodontitis: an immunohistochemical study.

CONTEXT: Periodontal disease is caused by chronic infection inducing an inflammatory reaction leading to breakdown of tooth-supporting tissues. There are various risk factors for the disease, and smoking is one of them. Apoptosis plays a critical role in the regulation of inflammation and host immune response which helps in tissue homeostasis, and a disturbance in this is often associated with disease. The imbalance between the apoptosis and proliferation in the periodontal tissue results in periodontal disease. Neutrophils play an important role in the defense mechanism and are the most abundant immune cells in gingival inflammatory infiltrate in patients suffering from periodontal disease. Neutrophil disorders are associated with rapid destruction of periodontal tissues. AIM: To study the influence of smoking on apoptosis of neutrophils by quantifying them in the gingival connective tissue of smoking and nonsmoking subjects suffering from chronic periodontitis. MATERIALS AND METHODS: Thirty gingival biopsies were harvested from 15 smoking and 15 nonsmoking subjects who suffered from chronic periodontitis. The apoptosis of neutrophils was assessed and quantified using p53 monoclonal mouse antihuman antibody. STATISTICAL ANALYSIS USED: Chi-square/Fisher's exact test was used to find the significance of study parameters on a categorical scale between the two groups. RESULTS: Neutrophil apoptosis was significantly more in the group of nonsmokers. There was no statistical difference between plaque and bleeding index, but there was a significant increase in clinical attachment loss among smokers. CONCLUSIONS: The study reveals that smoking plays a significant role in the inhibition of neutrophil apoptosis, thereby contributing to the destruction of periodontal tissues in periodontitis.