Milky spots in the greater omentum are predominant sites of local tumour cell proliferation and accumulation in the peritoneal cavity

The role that milky spots in the greater omentum play in tumour cell spread in the peritoneal cavity is presently not fully understood. To study whether intraperitoneally injected tumour cells appear preferentially in milky spots of the greater omentum and to study the changes in the greater omentum, and especially in the cell population of milky spots after tumour cell infiltration, the following study was performed. A detailed temporal sequences of changes in morphology and cellular composition in milky spots of the greater omentum of Wag/Rij rats 5, 15, 30, 60 min, 2, 4, 8, 16, 24 h, 2, 4, 8 days and 2 and 4 weeks after intraperitoneal administration of 2.0 × 10⁶ CC 531 tumour cells was investigated by light microscopy and electron microscopy (pre-embedding labelling). Our data showed that the milky spots in the greater omentum were the sites to which tumour cells migrated preferentially from the peritoneal cavity. The tumour cells infiltrated the milky spots and formed clusters within. The cellular population in milky spots reacted by a very rapid influx of young macrophages during the first hour and an increase of the total number of cells (P < 0.01). After 4 h tumour cells were also located on the greater omentum outside the area of the milky spots. Around these tumour cell deposits, new milky spots are formed, which increased the total number of milky spots. The cells present in milky spots are not capable of reversing the growth of tumours and finally a solid omental cake of tumour cells is formed. Received: 30 June 1998 / Accepted: 3 September 1998     

Macrophage colony-stimulating factor is indispensable for repopulation and differentiation of Kupffer cells but not for splenic red pulp macrophages in osteopetrotic (<Emphasis Type="Italic">op</Emphasis>/<Emphasis Type="Italic">op</Emphasis>) mice after macrophage depletion

We previously reported that macrophage colony-stimulating factor (M-CSF, CSF-1) played important roles in the process of the repopulation of Kupffer cells after their elimination by administration of liposome-entrapped dichloromethylene diphosphonate (lipo-MDP). In this study, we examined the repopulation of Kupffer cells and splenic red pulp macrophages in osteopetrotic (op/op) mice defective in the production of functional M-CSF and their littermate mice by using the lipo-MDP model. In untreated op/op mice, numbers of F4/80-positive Kupffer cells in the liver and F4/80-positive splenic red pulp macrophages were reduced. Repopulation of Kupffer cells and splenic macrophages was observed in littermate (op/+) mice liver by 14 days after depletion. However, in op/op mice, repopulation of Kupffer cells was not observed in Kupffer-cell-depleted op/op mice until 56 days after depletion, whereas splenic red pulp macrophages repopulated and recovered to the level of control op/op mice by 10 days after depletion. Single injection of M-CSF was effective for the induction of the repopulation of Kupffer cells, and daily administration of M-CSF induced remarkable repopulation and maturation of Kupffer cells and proliferation of macrophage precursor cells in the liver of Kupffer-cell-depleted op/op mice. These results suggest that Kupffer cells are completely M-CSF-dependent tissue macrophages, whereas splenic red pulp macrophages are composed of M-CSF-dependent macrophages and M-CSF-independent macrophages. This mouse model provides a useful tool for the study of effects of growth factor on Kupffer cell differentiation in vivo. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, NIH grant CA20408, and a Tsukada Memorial Grant (2000).     

Lymphoid and non-lymphoid cells in nasal-associated lymphoid tissue (NALT) in the rat

Summary Lymphocyte and macrophage subpopulations and the stroma of mucosa-associated lymphoid tissue in the nasal cavity of the rat were examined by application of immunohistochemical and enzyme histochemical methods to cryostat sections. Nasal-associated lymphoid tissue was composed of a loose reticular network with lymphocytes and macrophages, covered by epithelium. The epithelium was infiltrated with B cells, T helper (W3/13-positive) and T suppressor/cytotoxic or large granular cells (OX8-positive), ED1-positive macrophages and Ia-positive cells. The B cell areas were populated by B cells, immunopositive for surface IgM or IgG. B cells with surface IgA or IgE were rare. Germinal centres were found infrequently. T helper cells were scattered throughout the B cell area. A few ED1-positive macrophages and ED5-positive follicular dendritic cells were observed. Strong Ia staining (mostly of B cells) was found in this area. The T cell areas contained T helper and T suppressor/cytotoxic cells in about equal amounts, and numerous ED1-positive macrophages. ED1 staining was also found in the subepithelial area. Numerous ED1-, ED2- and ED3-positive macrophages were found in the border between the lymphoid mass and the surrounding connective tissue. A few non-lymphoid cells showed weak acid phosphatase or non-specific esterase activity. The morphological observations suggest that nasal-associated lymphoid tissue plays an important role in the first contact with inhaled antigens.     

The peritoneal cavity provides a protective niche for B1 and conventional B lymphocytes during anti-CD20 immunotherapy in mice

Although anti-CD20 immunotherapy effectively treats human lymphoma and autoimmune disease, the in vivo effect of immunotherapy on tissue B cells and their subsets is generally unknown. To address this, anti-mouse CD20 mAbs were used in a mouse model in which the extent and kinetics of tissue B cell depletion could be assessed in vivo. CD20 mAb treatment depleted most mature B cells within 2 days, with 95-98% of B cells in the bone marrow, blood, spleen, lymph nodes, and gut-associated lymphoid tissues depleted by day 7, including marginal zone and follicular B cells. The few spleen B cells remaining after CD20 mAb treatment included pre-B, immature, transitional, and some B1 B cells that expressed CD20 at low levels. By contrast, peritoneal cavity B cells expressed normal CD20 densities and were coated with CD20 mAb, but only 30-43% of B1 cells and 43-78% of B2 cells were depleted by day 7. Spleen B cells adoptively transferred into the peritoneal cavity were similarly resistant to mAb-induced depletion, while transferred B cells that had migrated to the spleen were depleted. However, peritoneal B1 and B2 cells were effectively depleted in mAb-treated wild-type and C3-deficient mice by thioglycolate-induced monocyte migration into this otherwise privileged niche. Inflammation-elicited effector cells did not promote peritoneal cavity B cell depletion in FcR-deficient mice treated with CD20 mAb. Thus, the majority of CD20(+) cells and B cell subsets within lymphoid tissues and the peritoneum could be depleted efficiently in vivo through Fc-dependent, but C-independent pathways during anti-CD20 immunotherapy.     

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.     

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.     

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

Background: 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). Methods: 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. Results: 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.Conclusion: 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.     

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.     

Apparent Reversion to Trophism of a Free Omental Graft: A Preliminary Report

In a previous paper we reported evidence which suggested that a free omental graft has trophic characteristics similar to those of the chorionic epithelium of the early embryo, through which it establishes arteriolar or larger connections with aorta, heart and pericardium within 10 days after it has been placed in contact with the denuded surfaces of each structure. The theory of omental trophism was tested by dropping pieces of omentum completely detached into the pleural and peritoneal cavities of white rats. When examined 25 or more days later a percentage of omental grafts appeared healthy and were attached to pleura, lung, heart and to various structures in the peritoneal cavity. In the pleural grafts, histological sections revealed a continuity between the omental grafts and the sub-pleural tissues. A blood-hungry free omental graft appears to seek out its own blood supply. Neither pleura nor serosa was able to repel its primitive probing for a new blood supply.     

Hepatocyte growth factor and macrophage-stimulating protein are upregulated during excisional wound repair in rats

Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP) are structurally related molecules that stimulate epithelial cell proliferation and migration. MSP also acts directly as a chemoattractant for resident macrophages. These activities are integral to the wound repair processes of inflammation, epithelialization and tissue remodelling. To begin to examine the involvement of HGF and MSP in healing of cutaneous wounds we have mapped the temporal expression of these two molecules and their receptors, MET and RON respectively, in adult rat excisional wounds. Four 2x2-cm full-thickness excisional wounds were created on the dorsum of 18 rats, and biopsies were taken through the wounds at 3, 5, 7, 14, 21, and 28 days postwounding. These biopsies were analyzed using immunofluorescent staining and in situ hybridization (ISH). The number of cells staining positively for HGF and MET significantly increased in response to wounding. HGF staining and mRNA peaked at 7 days postwounding whereas MET was upregulated earlier, peaking after 3 days. Both HGF and MET protein were observed in fibroblasts of the dermis and in the newly forming granulation tissue. ISH studies also revealed that fibroblasts at the wound edges and within the newly forming granulation tissue also expressed HGF and c-met mRNA. Immunofluorescent staining revealed both MSP and RON within the wound, with maximum staining occurring between 7 and 21 days for both the ligand and receptor. In addition, MSP co-localized with a small subset of ED1-positive cells (monocytes). In contrast, ED2-positive cells (macrophages) did not co-localize with MSP. Thus, increased expression of HGF, MSP and their receptors MET and RON respectively was observed in response to wounding. Furthermore, MSP co-localization with a subset of monocytes may confirm a role for MSP in the activation of mature macrophages, which may be important in tissue remodelling.     

Ontogeny of macrophage subpopulations and Ia-positive dendritic cells in pulmonary tissue of the rat

Summary The development of macrophage subpopulations and dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gutassociated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.Fellow of the Royal Netherlands Academy of Arts and Sciences     

Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate

Summary Rats received a single intravenous injection with liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP). This treatment resulted in the elimination of macrophages in spleen and liver within 2 days. Macrophages ingest the liposomes and are destroyed by the drug, which is released from the liposomes after disruption of the phospholipid bilayers under the influence of lysosomal phospholipases. Repopulation of macrophages in spleen and liver was studied at different time intervals after treatment. Macrophages in the liver (Kupffer cells) and red pulp macrophages in the spleen were the first cells to reappear, followed by marginal metallophilic macrophages and marginal-zone macrophages in the spleen. Different markers of the same cell did not reappear simultaneously. On the other hand, the same marker (recognized by the monoclonal antibody ED2) reappeared much more rapidly in the liver than in the spleen. The present results in the rat were different from those earlier obtained in the mouse. Red pulp macrophages were the first cells and marginal zone macrophages were the last cells to repopulate the spleen in both rodents after treatment with Cl2MDP liposomes. However, there was much more overlap in the repopulation kinetics of splenic macrophage subpopulations in the rat, when compared with the mouse.     

Characterization of Omental Immune Aggregates during Establishment of a Latent Gammaherpesvirus Infection

Herpesviruses are characterized by their ability to establish lifelong latent infection. The gammaherpesvirus subfamily is distinguished by lymphotropism, establishing and maintaining latent infection predominantly in B lymphocytes. Consequently, gammaherpesvirus pathogenesis is closely linked to normal B cell physiology. Murine gammaherpesvirus 68 (MHV68) pathogenesis in laboratory mice has been extensively studied as a model system to gain insights into the nature of gammaherpesvirus infection in B cells and their associated lymphoid compartments. In addition to B cells, MHV68 infection of macrophages contributes significantly to the frequency of viral genome-positive cells in the peritoneal cavity throughout latency. The omentum, a sheet of richly-vascularized adipose tissue, resides in the peritoneal cavity and contains clusters of immune cell aggregates termed milky spots. Although the value of the omentum in surgical wound-healing has long been appreciated, the unique properties of this tissue and its contribution to both innate and adaptive immunity have only recently been recognized. To determine whether the omentum plays a role in gammaherpesvirus pathogenesis we examined this site during early MHV68 infection and long-term latency. Following intraperitoneal infection, immune aggregates within the omentum expanded in size and number and contained virus-infected cells. Notably, a germinal-center B cell population appeared in the omentum of infected animals with earlier kinetics and greater magnitude than that observed in the spleen. Furthermore, the omentum harbored a stable frequency of viral genome-positive cells through early and into long-term latency, while removal of the omentum prior to infection resulted in a slight decrease in the establishment of splenic latency following intraperitoneal infection. These data provide the first evidence that the omentum is a site of chronic MHV68 infection that may contribute to the maintenance of chronic infection.     

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro

 Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i. p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage- colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i. p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth. Received: 11 April 1996 / Accepted: 21 May 1996     

Reticulum cells in the ontogeny of nasal-associated lymphoid tissue (NALT) in the rat

This study concerns the ontogeny of reticulum cells (RC) in the nasal-associated lymphoid tissue (NALT) of Wistar and Brown-Norway rats. A panel of monoclonal antibodies (mAb) directed against RC in peripheral lymphoid organs (antibodies ED10ED15) was used, together with a recently developed antibody ED17, which recognizes macrophages and Langerhans cells. Early in embryogenesis, staining with common connective tissue markers, ED14 and ED15, was found. ED17-positive cells were present before cells positive to ED1, a pan-macrophage marker, or Ia glycoproteins were observed. The first differentiation of reticulum was seen at the day of birth, when ED10 recognized a distinct area in the nasal mucosa. The first T-lymphocytes were found at the same time. Two days after birth, B-cells and ED11-positive cells were present in the NALT area. Fourteen days after birth, T- and B-cell compartments were recognizable. ED10 was found predominantly in the T-cell area and ED11 was mainly confined to the B-cell compartment. We conclude that the development of the NALT is closely accompanied by the phenotypic specialization of the reticulum. This suggests that the reticulum plays an important role in the compartmentalization of NALT tissue and in the retention of lymphocyte subsets within these compartments.     

The involvement of omentum and its milky spots in the dynamics of peritoneal macrophages

The investigation has been carried out on stimulated and unstimulated peritoneal cavities of rats. China ink and Corynebacterium parvum were injected i.p. both as peritoneal stimuli and markers. Omenta were picked up at time intervals beginning with 10 min and up to seven days after the i.p. injection. The light and electronmicroscopic investigation showed after 10-30 minutes labeled macrophages stuck as monolayers on some peritoneal areas corresponding to the milky spots which developed in size and number. Days after the i.p. injection the labeled macrophages were found deeper in the milky spots. After the fourth day they appeared in the regional lymph nodes. The milky spots contained also large lymphocytes and plasma cells. The results suggest that milky spots are not only places of resident macrophages development and release in the peritoneal cavity but also their exit pathways. Therefore the omentum leads the traffic of peritoneal macrophages. The developed milky spots play also the role of lymphoid structures providing grounds for macrophage-lymphocyte contacts.     

Origin and Kinetics of Resident Tissue Macrophages

Abstract To elucidate the origin and renewal kinetics of peritoneal macrophages, as a typical example of the mononuclear phagocytic system, syngeneic rats were treated with tritiated thymidine [³H]TdR and leucocytes were transferred to unlabelled recipients over a bilateral arteriovenous shunt. Labelled and unlabelled monocytes were evenly distributed in both animals as shown by autoradiography. It was ascertained that no ‘autoradiographically’ detectable reutilization of label occurred and that transferred cells showed undisturbed kinetics. the results imply: 1 resident peritoneal macrophages derive from blood monocytes; 2 peritoneal macrophages represent a homogeneous population in respect to their cellular origin; 3 blood monocytes as a myelogeneous cell line do not represent a generative end cell. They migrate into the tissue (peritoneal cavity) and differentiate into resident macrophages, undergoing on average one mitosis per cell during a period of approximately 7 days. 4 resident peritoneal macrophages are derived 50% from blood monocytes and 50% from division in situ; and 5 under steady-state conditions the renewal rate amounts to 0. 18%/h, which yields a half-life time of 16 days and a renewal time of 23 days.     

Suppression of macrophage infiltration into the conjunctiva by clodronate liposomes in experimental immune-mediated blepharoconjunctivitis

Macrophages infiltrate the conjunctiva in severe cases of allergic conjunctivitis (AC) such as atopic keratoconjunctivitis (AKC). We established experimental immune-mediated blepharoconjunctivitis (EC) in Brown Norway (BN) rats as a model for severe types of AC. We investigated whether macrophage infiltration in the conjunctiva in this EC model is inhibited by clodronate liposomes (CL2MDP-lip). The numbers of ED1-positive but not ED2-positive macrophages in the conjunctivas were increased by the induction of EC. Subconjunctival injection of CL2MDP-lip decreased the number of ED2-positive but not ED1-positive macrophages in the conjunctivas of naive rats. CL2MDP-lip did not affect macrophages in the spleen. Subconjunctival injection of CL2MDP-lip into EC-developing BN rats decreased the number of ED2-positive macrophages at all the time points. ED1-positive cell infiltration was inhibited when treatment was administered just prior to OVA challenge. Intravenous injection of CL2MDP-lip decreased the number of ED2-positive cells in the conjunctiva. Thus, we conclude that CL2MDP-lip inhibits infiltration of macrophages into the conjunctiva within 24 h of antigen challenge.     

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5⁺ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.