Omental Milky Spots Research Articles

The role of the CCL22-CCR4 axis in the metastasis of gastric cancer cells into omental milky spots.

BackgroundThe omentum is one of the initial sites for peritoneal metastasis because it possesses milky spots that provide a microenvironment for cancer cells to readily migrate and grow into micrometastases. This study investigated the role of the CCL22-CCR4 axis in gastric cancer cells selectively infiltrating into milky spots.MethodsGastric cancer MFC cells labelled with DiI were injected intraperitoneally into strain 615 mice. The mice were euthanised at specified intervals and the omentum was excised for immunohistochemistry. The effects of CCL22 on the proliferation and migration of MFCs were assessed by MTT and trans-well assays. RT-PCR and Western blot analysis detected CCR4 mRNA and protein expression levels in MFCs. Immunohistochemistry was used to analyse CCL22 and CCR4 expression in the milky spot micrometastases.ResultsTwo weeks after intraperitoneal injection, the milky spot areas were completely occupied by proliferating gastric cancer cells and cell cluster-type micrometastases were observed. In contrast, cancer cells formed single cell-type micrometastases in the non-milky spot areas. MFCs expressed CCR4, which was localised on the cell surface and or in the cytoplasm. Different concentrations of CCL22 significantly increased the proliferation ability of MFCs. Additionally, concentrations of CCL22 between 10–100 ng/ml significantly increased the migration of MFCs. Within omental milky spots, CCL22 was localised mainly on the cell surface and or in the cytoplasm. Within sections of omental milky spot micrometastases, CCR4 was recognised on or in gastric cancer cells, constituent cells milky spots, blood cells and blood endothelial cells.ConclusionsOmental milky spots are a congenial microenvironment for peritoneal free gastric cancer cells to migrate, survive, and establish cell cluster-type metastases. The CCL22-CCR4 axis contributes to this selective infiltration process.

Abstract B33: Omental milky spots serve as a niche for ovarian cancer metastatic colonization

Abstract Introduction: Metastatic ovarian cancer remains an urgent clinical problem. The homing and invasion of cancer cells into the omentum, the preferred site of ovarian cancer metastasis, is a rate-limiting step in disease progression. We have recently shown that immune cell-containing structures known as milky spots are required for colonization of the omental adipose. Experiments were designed using well-established ovarian cancer models and experimental metastasis assays to answer the following questions: Does cancer cell localization depend upon the immune composition of the milky spots? Do cancer cells utilize the native milky spot microenvironment, or does it undergo remodeling during colonization? Answers to these questions will be the foundation for mechanism-based studies aimed at identifying ovarian cancer-omental interactions that can be targeted therapeutically. Methods: Mouse strains with varying immune-competency were used to: 1) test the effect of immune composition on volume of milky spots within the omentum; 2) determine the requirement of specific immune cell types on ovarian cancer cell lodging and invasion into milky spots; and assess the effect of ovarian cancer cell lodging on milky spot composition. Strains included: C57BL/6 (immunocompetent), Athymic Nude (T cell deficient), Beige Nude (NK and T cell deficient), Rag1-/- (no mature T or B cells, no CD3+ or T cell receptor a-b positive cells), and Igh6-/- (lack mature B cells). Experimental metastasis assays used intraperitoneal injection of 1 x 106 ID8 (mouse) or SKOV3ip.1 (human) ovarian cancer cells into C57BL/6 mice, or immunodeficient mice, respectively. Milky spot volume in naïve omenta was calculated from digital whole-mounts constructed from Giemsa-stained tissue sections. Milky spot composition was evaluated using immunohistochemistry (IHC) for lymphocytes (CD45), T cells (CD3), B cells (B220), macrophages (F4/80), and endothelial cells (CD31). Results: ID8 and SKOV3ip.1 ovarian cancer cells localized to and proliferated in the milky spots. Studies using a panel of immune-deficient mice showed that the mouse genetic background does not alter omental milky spot number and size, nor does it affect ovarian cancer colonization. Analysis of digital whole mounts found no difference in the volume of milky spots in omenta from C57BL/6, Athymic Nude, Beige Nude, Rag1-/-, Igh6-/- mice. IHC of naïve omenta show the presence of B cells, T cells, and macrophages consistent with the genotype of the mouse. Abundant endothelial cells were present in milky spots corresponding to the dense vasculature that is a conserved feature of milky spots. Finally, evaluation of milky spot composition during metastatic colonization showed an influx of macrophages 24 hours and 3 days post injection (dpi). Staining for activated macrophages in metastases increased from week 1 to week 5; staining persisted until the experimental endpoint. Conclusions: Targeting the host tissue microenviroment is crucial to contain or reduce cancer spread, prolonging patient life. Use of genetic models ruled out a requirement of B, T, and NK cells in ovarian cancer cell homing to milky spots suggesting a role for macrophages in this process. A preliminary study indicates a temporal increase in activated macrophages upon cancer colonization. We speculate that cancer cells co-opt the normal developmental role of macrophages to promote their own growth. Work was supported by grants from the DOD (W81XWH-09-1-0127), NCI/NIH (2RO1CA089569), and Elsa U. Pardee Foundation; A Rivkin Center for Ovarian Cancer Research Pilot Study Award, and funds from the Section of Urology (The University of Chicago) Citation Format: Venkatesh Krishnan, Robert Clark, Michael Schoof, Irving Rodriguez, Marina Chekmareva, Carrie Rinker-Schaeffer. Omental milky spots serve as a niche for ovarian cancer metastatic colonization. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B33.

Abstract B65: Milky spots are required for ovarian cancer metastatic colonization of peritoneal adipose depots

Abstract Introduction: In ovarian cancer, attachment of cells to the omentum represents a rate-limiting step in metastasis formation. Thus, there is significant interest in identifying the tissue microenvironments involved in cancer cell colonization of this preferred metastatic site. The omentum is composed of translucent membranes and adipose, which contains immune structures known as milky spots. Two general models of omental colonization can be discerned from the published literature. In the “milky spot-driven” model, cancer cell lodging is due to factors produced by milky spots which serve as a colonization niche1. In contrast, the “adipose-driven” model proposes that adipocyte-derived factors are solely responsible for cancer cell lodging2. By taking advantage of peritoneal adipose deposits which either contain or lack milky spots, we set forth to test the hypothesis that these unusual immune structures are required for cancer cell colonization of adipose-rich tissues such as the omentum. Methods: Ovarian cancer cell migration, lodging and growth was assessed in a panel of peritoneal fat depots which either contain milky spots (omentum, splenoportal fat) or lack them (uterine fat, mesentery, and gonadal fat). The presence or absence of milky spots was confirmed by histology and immunohistochemistry (IHC) for CD45. Ovarian cancer cell lodging was evaluated via experimental metastasis assays using SKOV3ip.1 (human) or ID8 (mouse) ovarian cancer cells injected into Athymic Nude or C57BL/6 mice, respectively. At 7 days post injection (dpi) peritoneal fat depots were harvested and evaluated for the presence of cancer cells using histology and pan-cytokeratin IHC. The relative chemoattractant ability of tissue-conditioned media was evaluated by transwell migration assays using SKOV3ip.1 and ID8 cells. Results: The presence of milky spots in omental and splenoportal fat and their absence in uterine, gonadal, and mesenteric fat was confirmed by histology and IHC. In metastasis assays, cancer foci were observed in omental and splenoportal milky spots at 7 dpi. In contrast, cancer cells were never observed in adipose that lacked milky spots; even at 63 dpi. Consistent with this, conditioned media from milky-spot containing tissues had a significantly increased ability to promote ovarian cancer cells migration . Conclusions: In sum, our findings show that milky spots are an absolute requirement for cancer cell lodging on peritoneal adipose. Consistent with this, migration assays show that the presence of milky spots causes a significant enhancement in the ability of peritoneal adipose to promote directed migration. Taken together, these finding suggest that while adipose may secrete a general chemoattrative signal, additional contributions from the milky spots are required for localization and invasion. Current experiments include investigating the chemoattractant(s) used by the milky spots to attract cancer cells and the cell type(s) responsible for their secretion. Competing interests: The authors declare no competing interests.

Effects of high-fat diet on B1 B cells in obesity-associated inflammatory diseases (54.28)

Abstract Objective: High-fat diet (HFD)-induced abdominal obesity is a major risk factor for diabetes and atherosclerosis, yet the immunological mechanisms underlying these associations are still unclear. B1 B lymphocytes reside in the peritoneal cavity and omentum in direct contact with abdominal visceral adipose tissue. Their physiological location and functions make them an ideal intermediary for obesity-induced inflammatory disease. This study aimed to dissect the B1 B cell response to HFD-induced obesity in diabetes and atherosclerosis. Methods: gld/ApoE-/- mice fed a high-fat Western diet (WD) were used to model diet-accelerated atherosclerosis and autoimmunity. Wild type C57BL/6 mice fed a HFD were used to model diet-induced obesity and diabetes. Control mice from each group were fed with a low-fat control diet (CD). Cells from the peritoneal cavity and omentum were studied using flow cytometry. Hematoxylin/Eosin and immunohistochemistry stainings were performed on paraffin-embedded tissues. Results: Compared to their CD-fed counterparts, WD-fed gld/ApoE-/- mice exhibited enlarged omental milky spots and decreased peritoneal B1 cells. HFD-fed C57BL/6 mice demonstrated enlarged omental milky spots and reduced peritoneal B1 B cells compared to CD-fed counterparts. Conclusion: The similar B1 B cell response pattern to diet in both gld/ApoE-/- and WT mice indicates that HFD has a profound influence on the B1 B cell immune response regardless of genetic susceptibility.

LPS-Induced Migration of Peritoneal B-1 Cells is Associated with Upregulation of CXCR4 and Increased Migratory Sensitivity to CXCL12

B-1 cells, which constitute a predominant lymphocyte subset in serosal cavities and produce most of natural antibodies, are subdivided into the CD5+ B-1a and CD5- B-1b cell subpopulations, but the differential roles of B-1a and B-1b cells are not well understood. We report that B-1a cells preferentially migrate out of the peritoneal cavity and upregulate the expression of CXCR4 with heightened sensitivity to CXCL12 and CXCL13 upon LPS treatment compared to B-1b and B-2 cells. Whereas B-1a cells were slightly more abundant than B-1b and B-2 cells in the homeostatic condition, the number of B-1a cells preferentially decreased 48 hr after LPS treatment. The decrease in the peritoneal B-1a cell number was accompanied with increased migration of B-1a cells toward CXCL-12 and CXCL-13 in in vitro transmigration assay using peritoneal B cells from LPS treated mice. The expression level of CXCR4, but not of CXCR5, was also more prominently increased in B-1a cells upon LPS stimulation. LPS-stimulated B-1a cells did not accumulate in omental milky spots in contrast to B-2 cells. These results suggest that B-1a cells actively migrate out of the peritoneal cavity through the regulation of the migratory responsiveness to chemokines and actively participate in systemic immune responses.

Omental milky spots in screening gastric cancer stem cells

The existence of cancer stem and progenitor cells in solid tumors has been widely postulated. However, neither the cancer stem cells nor the cancer progenitor cells have been definitively identified and functionally characterized. Here we propose a new strategy to identify and isolate gastric cancer stem cells -using omental milky spots to screen gastric cancer stem cells in peritoneal metastasis mouse models of gastric cancer. In this study, we used the property that the macrophages in omental milky spots are cytotoxic against tumor cells and so able to screen and collect cancer stem cells. Our findings suggest that macrophages in omental milky spots have not only cytotoxic properties against tumor cells but also provide a microenvironment within milky spots in which cancer stem cells are capable to survive and grow into micrometastasis. Omental milky spot become a cancer stem cell niche in this situation. Further we studied the omental milky spots for screening gastric cancer cells (OMSS-GCCs) and found that omental milky spot enriched the volume of gastric cancer stem cells. Tumors were consistently generated after an injection of 1×103 OMSS-GCCs. OMSS-GCCs high express CD133 and low express CD324. Omental milky spots are a highly efficient "natural filter" for screening gastric cancer stem cells.

Omental and pleural milky spots: different reactivity patterns in mice infected with Schistosoma mansoni reveals coelomic compartmentalisation

In vertebrate animals, pleural and peritoneal cavities are repositories of milky spots (MS), which constitute an organised coelom-associated lymphomyeloid tissue that is intensively activated by Schistosoma mansoni infection. This study compared the reactive patterns of peritoneal MS to pleural MS and concluded from histological analysis that they represent independent responsive compartments. Whole omentum, lungs and the entire mediastinum of 54 S. mansoni-infected mice were studied morphologically. The omental MS of infected animals were highly activated, modulating from myeloid-lymphocytic (60 days of infection) to lymphomyeloid (90 days of infection) and lymphocytic or lymphoplasmacytic (160 days of infection) types. The non-lymphoid component predominated in the acute phase of infection and was expressed by monocytopoietic, eosinopoietic and neutropoietic foci, with isolated megakaryocytes and small foci of late normoblasts and mast cells. Nevertheless, pleural or thoracic MS of infected mice were monotonous, consisting of small and medium lymphocytes with few mast and plasma cells and no myeloid component. Our data indicate that compartmentalisation of the MS response is dependent on the lymphatic vascularisation of each coelomic cavity, limiting the effects or consequences of any stimulating or aggressive agents, as is the case with S. mansoni infection.

Targeted delivery of oligomannose‐coated liposome to the omental micrometastasis by peritoneal macrophages from patients with gastric cancer

We recently developed a novel drug delivery system (DDS) using oligomannose-coated liposomes (OMLs), which are effectively taken up by mouse peritoneal macrophages to carry anticancer drugs to omental milky spots known as initial metastatic sites in the peritoneal cavity in mice. However, the feasibility of the clinical application of this DDS to gastric cancer patients remains essentially unknown. In the present study, we investigated whether human peripheral blood monocytes (PBMs) and human peritoneal macrophages (PEMs) could successfully uptake OMLs and deliver them to the micrometastatic foci in the mouse omentum and resected omentum from cancer patients ex vivo. When OMLs were incubated with the PBMs from four healthy volunteers in vitro, an average 88% of CD14-positive PBMs, most of which also express CD206, took up OMLs, and this uptake was significantly inhibited by alpha-methylmannoside. In the experiment using PEMs obtained from peritoneal washes of five gastric cancer patients, the average uptake rate (63%) of OML by CD14-positive PEMs was somewhat lower than that of PBMs, but in three advanced gastric cancer patients the uptake rate of OMLs was 76% which was comparable to that of mouse PEMs. Oligomannose-coated liposome (OML)-incorporated PBMs and PEMs were successfully accumulated at the micrometastatic foci at the omentum formed after intraperitoneal injection of GFP-tagged gastric cancer cells into mice. Furthermore, OML-incorporated PBMs substantially accumulated to tumor foci in the surgically resected human omentum ex vivo. These results suggest that OMLs using human monocytes/macrophages as a cellular vehicle have the potential to target peritoneal micrometastasis in the omentum of gastric cancer patients.

Omental milky spots – highly efficient “natural filter” for screening gastric cancer stem cells

Over the last decades, cancer stem cells (CSCs) have been identified in different solid tumors and three strategies have been employed to identify them as follows: (1) expression of cell surface markers, (2) ability to exclude fluorescent dye Hoescht 33342 in a cell population termed “side population” (SP), and (3) formation of tumorspheres in suspension culture. But these methods focus on a minor subset of tumor cells, initial CSCs are still hidden among them and genuine CSCs have not yet been isolated and purified. Here we propose a new strategy to identify and isolate gastric cancer stem cells – using omental milky spots to screen gastric cancer stem cells in peritoneal metastasis mouse models of gastric cancer. This strategy directly target cancer stem cells––which can cause a new tumor by itself. Omental milky spots and their macrophages become a highly efficient “natural filter” for screening gastric cancer stem cells.

Paracrine Provision of Lipids in the Immune System

Adipocytes enclosing lymph nodes (and omental milky spots) are specialized for paracrine interactions with lymphoid cells. They secrete and respond to cytokines and their lipids contain more polyunsaturated fatty acids, precursors of eicosanoids and docosanoids. Dendritic cells and lymph node lymphoid cells obtain many, possibly all, fatty acids found in structural lipids from adjacent perinodal adipocytes. By supplying appropriate fatty acids to lymph nodes and dendritic cells, perinodal adipocytes intervene between the diet and the immune systems nutrition and partially emancipate it from fluctuations in abundance and composition of dietary lipids. Site-specific differences in the composition of lipids taken up from contiguous adipocytes create further dendritic cell diversity. Local immune stimulation induces the formation of more adipocytes, which may contribute to mesenteric and omental hypertrophy in chronic inflammatory diseases such as HIV-infection. Perinodal adipose tissue is abnormal in Crohns disease, leading to insufficient fatty acid precursors of eicosanoids in mesenteric lymph nodes, even when they are plentiful in the diet. Perivascular, epicardial and perinodal adipose tissue have some common properties. Adipose tissue partitioned into many depots, some specialised for paracrine interactions, is a fundamental feature of mammals, underpinning multiple simultaneous responses to pathogens and more efficient utilization of lipids. Keywords: HIV-associated adipose redistribution syndrome, adipose tissue, perinodal adipocytes, lymph nodes, dendritic cells, nutrition, Crohn's disease

Omental Milky Spots Develop in the Absence of Lymphoid Tissue-Inducer Cells and Support B and T Cell Responses to Peritoneal Antigens

The omentum is a site of B1 cell lymphopoiesis and immune responsiveness to T cell-independent antigens. However, it is unknown whether it supports immune responses independently of conventional lymphoid organs. We showed that the omentum collected antigens and cells from the peritoneal cavity and supported T cell-dependent B cell responses, including isotype switching, somatic hypermutation, and limited affinity maturation, despite the lack of identifiable follicular dendritic cells. The omentum also supported CD4+ and CD8+ T cell responses to peritoneal antigens and recruited effector T cells primed in other locations. Unlike conventional lymphoid organs, milky spots in the omentum developed in the absence of lymphoid tissue-inducer cells, but required the chemokine CXCL13. Although the lymphoid architecture of milky spots was disrupted in lymphotoxin-deficient mice, normal architecture was restored by reconstitution with lymphotoxin-sufficient hematopoietic cells. These results indicate that the milky spots of the omentum function as unique secondary lymphoid organs that promote immunity to peritoneal antigens.

Differential Molecular and Anatomical Basis for B Cell Migration into the Peritoneal Cavity and Omental Milky Spots

The constitutive migration of B cells from the circulation into the peritoneal cavity and back is essential for peritoneal B cell homeostasis and function. However, the molecular machinery and the anatomical basis for these migratory processes have hardly been investigated. In this study, we analyze the role of integrins as well as the role of the omentum for B2 cell migration into and out of the peritoneal cavity of mice. We demonstrate that alpha(4)beta(7) integrin-mucosal addressin cell adhesion molecule 1 interaction enables B2 cell migration from the circulation into omental milky spots but not into the peritoneum. In contrast, alpha(4)beta(1) integrin mediates direct entry of B2 cells into the peritoneal cavity as well as their retention at that site, limiting B2 cell egress via the draining parathymic lymph nodes. Surgical removal of the omentum results in a 40% reduced immigration of B2 cells from the circulation into the peritoneum but does not impair B cell exit from this compartment. In conclusion, these data reveal the existence of alternative routes for B2 cell entry into the peritoneal cavity and identify integrins as key factors for peritoneal B2 cell homeostasis, mediating B2 cell migration into and out of the peritoneal cavity as well as their retention at this site.

Insufficient ability of omental milky spots to prevent peritoneal tumor outgrowth supports omentectomy in minimal residual disease.

The greater omentum is frequently involved in the course of gastrointestinal and ovarian tumors. Therefore, common practice in surgical treatment for especially gastric and ovarian cancer includes removal of the greater omentum. Paradoxically, many immune cells, such as macrophages that accumulate in so-called milky spots, reside within the omentum and are cytotoxic against tumor cells ex vivo. Consequently, omental macrophages might play an important role in killing tumor cells, and may hereby prevent development into local peritoneal recurrences. In the present study, we therefore evaluated the role of the omentum and the clinical relevance of omentectomy in minimal residual disease (MRD). Tumor cell dissemination patterns on the omentum in a rat model were examined using DiI-labelled CC531s tumor cells. Additionally, intra peritoneal (i.p.) tumor load was investigated in rats that underwent omentectomy or sham laparotomy followed by i.p. injection of CC531s cells on day 21, which represented MRD. At 4 h post injection, tumor cells predominantly adhered on milky spots. Number of cells thereafter declined rapidly suggesting initial tumor killing functions in these specific immune aggregates. Despite initial reduction observed in milky spots, numbers of tumor cells however increased at fatty tissue stripes that border the omentum. This indicated proliferation at these locations, which corresponded to macroscopic observations of the omenta on day 21 after tumor cell injection. Omentectomy resulted in reduced intra-abdominal tumor load, which was completely attributable to the absence of the omentum, as tumor development did not differ on other sites. Even in the MRD group microscopic clusters of tumor cells located in the omentum eventually developed into macroscopic nodules. Since the ability of omental milky spots is, even in MRD, insufficient to prevent intra abdominal tumor outgrowth, omentectomy, which reduces tumor load, is recommended in surgical treatment of intra abdominal tumors that are prone to disseminate intraperitoneally.

Adipose tissue and the immune system

Adipocytes anatomically associated with lymph nodes (and omental milky spots) have many special properties including fatty acid composition and the control of lipolysis that equip them to interact locally with lymphoid cells. Lymph node lymphocytes and tissue dendritic cells acquire their fatty acids from the contiguous adipocytes. Lymph node-derived dendritic cells suppress lipolysis in perinodal adipocytes but those that permeate the adipose tissue stimulate lipolysis, especially after minor, local immune stimulation. Inflammation alters the composition of fatty acids incorporated into dendritic cells, and that of node-containing adipose tissue, counteracting the effects of dietary lipids. Thus these specialised adipocytes partially emancipate the immune system from fluctuations in the abundance and composition of dietary lipids. Prolonged, low-level immune stimulation induces the local formation of more adipocytes, especially adjacent to the inflamed lymph node. This mechanism may contribute to hypertrophy of the mesentery and omentum in chronic inflammatory diseases such as HIV-infection, and in smokers. Paracrine interactions between adipose and lymphoid tissues are enhanced by diets rich in n-6 fatty acids and attentuated by fish oils. The latter improve immune function and body conformation in animals and people. The partitioning of adipose tissue in many depots, some specialised for local, paracrine interactions with other tissues, is a fundamental feature of mammals.

Omental Milky Spots and Peritoneal Dialysis — Review and Personal Experience

Little research has been dedicated to milky spots (MS), except for their role in oncology. In the field of peritoneal dialysis (PD), studying MS could help in understanding peritoneal defenses. We reviewed the methods for detecting and counting omental MS and studied modifications induced by chemical and inflammatory stimuli. The reactions of MS to peritoneal catheters, PD solutions, and infection were studied in 32 rabbits. We also evaluated changes in MS in 39 serial omental biopsies from 16 patients with different histories of PD, and examined peritoneal biopsies from 38 patients with sclerosing peritonitis. The catheter provoked an immediate increase in the number and size of MS in rabbits. Intraperitoneal infusion of commercial PD solution containing 1.38% or 3.86% glucose for 30 days led to a slight but significant increase in the number and size of MS, without differences between the two glucose concentrations. Peritonitis caused a sharp increase in the number of MS in rabbits and humans, and a particular transformation. In patients with simple sclerosis, we observed normal MS having the same number and size as in patients without simple sclerosis. A few MS were found in only 2 patients with sclerosing peritonitis. Peritoneal dialysis activates omental MS. Peritoneal infection leads to a marked increase in the activity of MS, some of which undergo a singular transformation, casting doubt on previous theories about differentiation of MS from other lymphatic organs. Comparison with oncological studies indicates certain contact points. The presence of MS in PD patients with simple sclerosis is in contradiction to other morphological studies sustaining that MS act only when in contact with a fenestrated mesothelial basement membrane. Finally, the shortage of MS in patients with sclerosing peritonitis raises certain questions about etiopathogenesis.

Better Preservation of the Peritoneum in Rats Exposed to Amino Acid-Based Peritoneal Dialysis Fluid

Glucose-containing peritoneal dialysis fluids (PDF) show impaired biocompatibility, which is related partly to their high glucose content, presence of glucose degradation products, low pH, and lactate buffer, or a combination of these factors. In a rat chronic peritoneal exposure model, we compared effects of an amino acid-based PDF (AA-PDF) with a glucose-containing PDF on the peritoneal microcirculation and morphology. Two groups of rats received 10 mL of either fluid daily for 5 weeks via peritoneal catheters connected to implanted subcutaneous mini vascular access ports. Leukocyte-endothelium interactions in the mesenteric venules were investigated by intravital microscopy. Quantification of angiogenesis and fibrosis and inspection of the mesothelial cell layer were performed by light and electron microscopy. Daily exposure to glucose-containing PDF resulted in a significant increase in the number of rolling leukocytes in mesenteric venules, whereas instillation of AA-PDF did not change the level of leukocyte rolling. Glucose-containing PDF evoked a significantly higher number of milky spots in the omentum, whereas this response was significantly reduced in animals exposed to the AA-PDF (p < 0.02). Chronic instillation of glucose-containing PDF induced angiogenesis in various peritoneal tissues, accompanied by fibrosis in the mesentery and parietal peritoneum. Quantitative morphometric evaluation of omentum and mesentery showed a clear trend toward less angiogenesis after treatment with the AA-PDF compared to the glucose-containing PDF, which reached statistical significance in the parietal peritoneum (p < 0.04). Instillation of AA-PDF resulted in approximately 50% reduction of fibrosis in the mesentery (p < 0.04) and approximately 25% reduction in the parietal peritoneum (p < 0.009) compared to glucose-containing PDF. Glucose-containing PDF damaged the mesothelial cell layer, whereas the mesotheium was intact after AA-PDF treatment, as evidenced by electron microscopy. Our data in a rat chronic peritoneal exposure model clearly demonstrate reduced immune activation (evidenced by decreased number of rolling leukocytes and decreased induction of omental milky spots) and reduced neoangiogenesis, fibrosis, and mesothelial damage of the peritoneal membrane after treatment with AA-PDF compared to glucose-containing PDF.

Effect of Intraperitoneal Neutrophils Induced by OK432 on Malignant Ascites

To evaluate the efficacy of a streptococcal preparation, OK432, on malignant ascites in mice. PC-C203U (PC203) is a preparation of another strain of the streptococcal family, with the lowest antineoplastic action. To examine the survival curves of mice after the inoculation of BAMC-1 tumor cells, we gave intraperitoneal OK432, PC203, or saline as a control. Intraperitonal neutrophils were counted by cytospin, and interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and macrophage inflammatory protein (MIP)-1alpha were measured by enzyme-linked immunosorbent assay, and 8 h after the administration of OK432, PC203, and saline. Using electron microscopy, we examined the greater omental milky spots, where the in situ proliferation of neutrophils or macrophages takes place. The OK432 group had the best survival and the control group the worst. The ratio of intraperitoneal neutrophils to BAMC-1 was highest in the OK432 group and lowest in the control group. Quantitative IL-1beta, IL-6 and MIP-1alpha levels were correlated closely with survival. Electron microscopic examination of the milky spots revealed massive proliferation of neutrophils in the OK432 group, but not in the PC203 or control groups. OK432 effectively activated intraperitoneal neutrophils and a series of immunological chain reactions through an increase in IL-1beta, IL-6, and MIP-1alpha levels. Milky spots could have important antitumor effects in terms of the spread of neutrophils.

Paracrine interactions of mammalian adipose tissue.

Adipose tissue develops in and/or around most lymphoid tissues in mammals and birds. Early reports of this widespread association and hypotheses for its functional basis were long ignored in the planning of in vitro studies and the interpretation of in vivo results. Biochemical studies on rodent tissues reveal many site-specific properties of adipocytes anatomically associated with lymph nodes and omental milky spots that equip them to interact locally with lymphoid cells. The paracrine interactions are strongest for the most readily activated lymph nodes and are modulated by dietary lipids. Perinodal adipocytes contribute less than those in the large nodeless depots to whole-body lipid supplies during fasting. Observations on wild animals show that perinodal adipose tissue is selectively conserved even in starvation but does not enlarge greatly in natural obesity. Such paracrine provisioning of peripheral immune responses improves their efficiency and emancipates activated lymphocytes from competition with other tissues for blood-borne nutrients. The relationship is found in extant protherians and metatherians, so it almost certainly arose early in the evolution of mammals, possibly as part of the metabolic reorganisation associated with homeothermy, viviparity, and lactation. Prolonged disruption to paracrine interactions between lymphoid and adipose tissue may contribute to the HIV-associated adipose redistribution syndrome, causing selective hypertrophy of the mesentery, omentum, and other adipose depots that contain much activated lymphoid tissue. Skeletal and cardiac muscle may also have paracrine relationships with anatomically associated adipose tissue, but interactions between contiguous tissues have not been demonstrated directly.

Paracrine relationships between adipose and lymphoid tissues: implications for the mechanism of HIV-associated adipose redistribution syndrome

The adipocytes anatomically associated with lymph nodes and omental milky spots have site-specific properties that equip them to interact locally with lymphoid cells. Paracrine provisioning of peripheral immune responses improves their efficiency and emancipates activated lymphocytes from competition with other tissues for blood-borne nutrients. Prolonged disruption to such paracrine interactions might contribute to the HIV-associated adipose redistribution syndrome, causing selective hypertrophy of the mesentery, omentum and other lymphoid tissue-containing adipose depots, while nodeless depots atrophy.

Biodefense function of omental milky spots through cell adhesion molecules and leukocyte proliferation.

To evaluate the immunological functions of the greater omentum in the peritoneal cavity, the localization of cell adhesion molecules (CAMs) on mesothelial cells and leukocytes in the omental milky spots were studied in normal and lipopolysaccharide (LPS)-stimulated mice by means of immunoelectron microscopy. The milky spots featured numerous leukocytes among the dome-shaped mesothelial cells, even in the normal stable state. Leukocyte integrins LFA-1, Mac-1, and VLA-4 were preferentially localized to microvilli and ruffles of macrophages and lymphocytes. The mesothelial cells of the milky spots showed higher ICAM-1 levels than did those of other omental regions, and fibronectin was detected in the stomata. The number of leukocytes markedly increased following an increase in proliferating cell nuclear antigen (PCNA)-positive cells in the milky spots after LPS stimulation. The mesothelial cells contained VCAM-1 newly restricted to the microvilli and increasing amounts of ICAM-1. These results show that the omental milky spots are active sites for leukocyte migration and peritoneal leukocyte supply because of the presence of adhesion molecules and active cell proliferation. Proliferative active leukocytes and those that have migrated from vessels pass through the stomata via an interaction of VLA-4 and fibronectin, adhere to the microvilli of the activated mesothelial cell surface as the result of an interaction between ICAM-1/VCAM-1 and integrins, and exude into the peritoneal cavity. Much of the exudation and adhesion of leukocytes seen in the milky spots of LPS-stimulated mice may be attributable to an increase in cell proliferation and in the amounts of ICAM-1 and VCAM-1.