Thymic Macrophages Research Articles

Culture of Mouse Thymic Epithelial Cells in Serum-Free Medium.

Primary cell culture systems are widely used as a valuable method for analyzing the biological functions of specific cells in vitro. Recently, various serum-free primary cell culture methods have been developed that do not involve the use of animal serums. Since the thymus is comprised of many cell types, such as thymocytes, thymic epithelial cells, macrophages, and fibroblasts, thymic epithelial cells must be isolated for their functional analysis in vitro. This chapter describes the detailed protocol for the selective primary culture of thymic epithelial cells using defined serum-free medium.

Response of CD68-Positive Thymic Cells in Rats to Selenium and Carcinogen Administration

Macrophages are the key thymic cells that take part in the antigen-independent differentiation of T-lymphocytes and their further selection. The level of macrophages indicates changes in the functions of the organ under study. As an essential trace element, selenium is part of a variety of proteins and enzymes, which perform cytoprotective, antimutagenic, and anticancerogenic action. Selenium also plays a significant role in the functioning of the immune system, increasing the activity of natural killers, production of interleukins, and stimulation of phagocytosis. This study was aimed at investigating the response of thymic macrophages to the administration of selenium and a chemical carcinogen. During the experiment, an increase in the level of CD68-positive cells was revealed in all experimental animals receiving selenium and a carcinogen, as well as under their combined action. The observed effects were assumed to be related to the immune-stimulating effect of selenium.

Chemotherapy-Induced Thymic Involution: An Ultrastructural Study

Abstract Endogenous thymic repair is quite complex, demanding coordinated thymocyte and thymic stromal responses for T cell development and establishment of central tolerance. While the importance of thymic stroma, notably cortical (cTEC) and medullary (mTEC) thymic epithelial cells, has been shown in gain- and loss-of-function studies, endogenous thymic epithelial repair has never been resolved ultrastructurally. Using Transmission Electron Microscopy (TEM), we analyzed morphometrically thymi in mice receiving Cyclophosphamide (CTX). In vehicle-treated mice, the cortex was populous with thymocytes neighboring cTEC, which extended long cytoplasmic processes into a dendritic meshwork, called “cytoreticulum”. In CTX-treated mice though, thymocytes were scarcely met in the cortex and TEC appeared more circular/ellipsoid. The cytoreticulum was evidently collapsed, increasing total contact surface area among cTEC/mTEC subsets. Despite that thymic macrophages primarily mediate clearance of thymocytes failing positive/negative selection, those in CTX-treated thymi were rich in secondary lysosomes and many were found phagocytosing TEC. The surviving TEC in CTX-treated mice had increased autophagolysosomes per surface area unit of TEC cytoplasm compared to vehicle-treated ones. Analysis via intensity thresholding revealed higher electron density of enclosed particles, consistent with the presence of partly-digested amorphous material, indicating membranous organelle self-digestion (i.e., stress macroautophagy). These data propose an emerging hypothesis that survival of thymic epithelium following cytotoxic insult is mediated via a collective stress response involving macroautophagy activation in TEC. Funded by: 1)AAI careers in immunology Fellowship - trainee 2)new investigator's start up funds (PI)

PFKFB2-mediated glycolysis promotes lactate-driven continual efferocytosis by macrophages.

Resolving-type macrophages prevent chronic inflammation by clearing apoptotic cells through efferocytosis. These macrophages are thought to rely mainly on oxidative phosphorylation, but emerging evidence suggests a possible link between efferocytosis and glycolysis. To gain further insight into this issue, we investigated molecular-cellular mechanisms involved in efferocytosis-induced macrophage glycolysis and its consequences. We found that efferocytosis promotes a transient increase in macrophage glycolysis that is dependent on rapid activation of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), which distinguishes this process from glycolysis in pro-inflammatory macrophages. Mice transplanted with activation-defective PFKFB2 bone marrow and then subjected to dexamethasone-induced thymocyte apoptosis exhibit impaired thymic efferocytosis, increased thymic necrosis, and lower expression of the efferocytosis receptors MerTK and LRP1 on thymic macrophages compared with wild-type control mice. In vitro mechanistic studies revealed that glycolysis stimulated by the uptake of a first apoptotic cell promotes continual efferocytosis through lactate-mediated upregulation of MerTK and LRP1. Thus, efferocytosis-induced macrophage glycolysis represents a unique metabolic process that sustains continual efferocytosis in a lactate-dependent manner. The differentiation of this process from inflammatory macrophage glycolysis raises the possibility that it could be therapeutically enhanced to promote efferocytosis and resolution in chronic inflammatory diseases.

Central Role of Macrophages and Nucleic Acid Release in Myasthenia Gravis Thymus.

Myasthenia gravis (MG) is a neuromuscular disease mediated by antibodies against the acetylcholine receptor (AChR). The thymus plays a primary role in AChR-MG and is characterized by a type I interferon (IFN) signature linked to IFN-β. We investigated if AChR-MG was characterized by an IFN-I signature in the blood, and further investigated the chronic thymic IFN-I signature. Serum levels of IFN-β and IFN-α subtypes, and mRNA expression for IFN-I subtypes and IFN-stimulated genes in peripheral mononuclear blood cells (PBMCs) were analyzed. The contribution of endogenous nucleic acids in thymic expression of IFN-I subtypes was investigated in human thymic epithelial cell cultures and the mouse thymus. By immunohistochemistry, thymic CD68+ and CD163+ macrophages were analyzed in AChR-MG. To investigate the impact of a decrease in thymic macrophages, mice were treated with an anti-CSF1R antibody. No IFN-I signature was observed in the periphery emphasizing that the IFN-I signature is restricted to the MG thymus. Molecules mimicking endogenous dsDNA signalization (Poly(dA:dT) and 2'3'-cGAMP), or dexamethasone-induced necrotic thymocytes increased IFN-β and α-AChR expression by thymic epithelial cells, and in the mouse thymus. A significant decrease in thymic macrophages was demonstrated in AChR-MG. In mice, a decrease in thymic macrophages led to an increase of necrotic thymocytes associated with IFN-β and α-AChR expression. These results suggest that the decrease of thymic macrophages in AChR-MG impairs the elimination of apoptotic thymocytes favoring the release of endogenous nucleic acids from necrotic thymocytes. In this inflammatory context, thymic epithelial cells may overexpress IFN-β, which specifically induces α-AChR, resulting in self-sensitization and thymic changes leading to AChR-MG. ANN NEUROL 2023;93:643-654.

Thymic macrophages consist of two populations with distinct localization and origin.

Tissue-resident macrophages are essential to protect from pathogen invasion and maintain organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and defined their expression signature. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor SpiC with these cells. Single-cell RNA sequencing showed that the macrophages in the adult thymus are composed of two populations distinguished by the expression of Timd4 and Cx3cr1. Remarkably, Timd4+ cells were located in the cortex, while Cx3cr1+ macrophages were restricted to the medulla and the cortico-medullary junction. Using shield chimeras, transplantation of embryonic thymuses, and genetic fate mapping, we found that the two populations have distinct origins. Timd4+ thymic macrophages are of embryonic origin, while Cx3cr1+ macrophages are derived from adult hematopoietic stem cells. Aging has a profound effect on the macrophages in the thymus. Timd4+ cells underwent gradual attrition, while Cx3cr1+ cells slowly accumulated with age and, in older mice, were the dominant macrophage population in the thymus. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.

The TAM receptor tyrosine kinases Axl and Mer drive the maintenance of highly phagocytic macrophages.

Many apoptotic thymocytes are generated during the course of T cell selection in the thymus, yet the machinery through which these dead cells are recognized and phagocytically cleared is incompletely understood. We found that the TAM receptor tyrosine kinases Axl and Mer, which are co-expressed by a specialized set of phagocytic thymic macrophages, are essential components of this machinery. Mutant mice lacking Axl and Mer exhibited a marked accumulation of apoptotic cells during the time that autoreactive and nonreactive thymocytes normally die. Unexpectedly, these double mutants also displayed a profound deficit in the total number of highly phagocytic macrophages in the thymus, and concomitantly exhibited diminished expression of TIM-4, CD163, and other non-TAM phagocytic engulfment systems in the macrophages that remained. Importantly, these previously unrecognized deficits were not confined to the thymus, as they were also evident in the spleen and bone marrow. They had pleiotropic consequences for the double mutants, also previously unrecognized, which included dysregulation of hemoglobin turnover and iron metabolism leading to anemia.

Multiomics reveal the central role of pentose phosphate pathway in resident thymic macrophages to cope with efferocytosis-associated stress.

Tissue-resident macrophages (TRMs) are heterogeneous cell populations found throughout the body. Depending on their location, they perform diverse functions maintaining tissue homeostasis and providing immune surveillance. To survive and function within, TRMs adapt metabolically to the distinct microenvironments. However, little is known about the metabolic signatures of TRMs. The thymus provides a nurturing milieu for developing thymocytes yet efficiently removes those that fail the selection, relying on the resident thymic macrophages (TMφs). This study harnesses multiomics analyses to characterize TMφs and unveils their metabolic features. We find that the pentose phosphate pathway (PPP) is preferentially activated in TMφs, responding to the reduction-oxidation demands associated with the efferocytosis of dying thymocytes. The blockade of PPP in Mφs leads to decreased efferocytosis, which can be rescued by reactive oxygen species (ROS) scavengers. Our study reveals the key role of the PPP in TMφs and underscores the importance of metabolic adaptation in supporting Mφ efferocytosis.

Neurotransmitters Modulate Intrathymic T-cell Development.

The existence of a crosstalk between the nervous and immune systems is well established. Neurotransmitters can be produced by immune cells, whereas cytokines can be secreted by cells of nervous tissues. Additionally, cells of both systems express the corresponding receptors. Herein, we discuss the thymus as a paradigm for studies on the neuroimmune network. The thymus is a primary lymphoid organ responsible for the maturation of T lymphocytes. Intrathymic T-cell development is mostly controlled by the thymic microenvironment, formed by thymic epithelial cells (TEC), dendritic cells, macrophages, and fibroblasts. Developing thymocytes and microenvironmental cells can be influenced by exogenous and endogenous stimuli; neurotransmitters are among the endogenous molecules. Norepinephrine is secreted at nerve endings in the thymus, but are also produced by thymic cells, being involved in controlling thymocyte death. Thymocytes and TEC express acetylcholine receptors, but the cognate neurotransmitter seems to be produced and released by lymphoid and microenvironmental cells, not by nerve endings. Evidence indicates that, among others, TECs also produce serotonin and dopamine, as well as somatostatin, substance P, vasoactive intestinal peptide (VIP) and the typical pituitary neurohormones, oxytocin and arg-vasopressin. Although functional data of these molecules in the thymus are scarce, they are likely involved in intrathymic T cell development, as exemplified by somatostatin, which inhibits thymocyte proliferation, differentiation, migration and cytokine production. Overall, intrathymic neuroimmune interactions include various neurotransmitters, most of them of non-neuronal origin, and that should be placed as further physiological players in the general process of T-cell development.

Isolation and characterization of a novel antimicrobial compound from sub-aerial cyanobacterium Fischerella sp., isolated from building facades

This work was aimed at to extract a novel antimicrobial compound, especially against certain human pathogens, from a sub-aerial cyanobacterium Fischerella sp., isolated from the building facades of Bhubaneswar, Odisha (India). The antimicrobial activity was determined in different solvents and aqueous extracts using agar well diffusion method which were purified using column chromatography and TLC (twice), followed by direct bioautography, MIC, cytotoxicity and was characterized using UV-Vis, FTIR and LC-MS spectroscopic methods. The lowest MIC value (0.312 and 0.625 mg mL-1) was depicted by the acetone extracted single compound against Escherichia coli and Candida albicans. The bioautography and spectroscopic analysis revealed the compound to be an alkaloid which was identified as pentaphenyl ferrocene carboxamide and found to be non-toxic against Catla thymus macrophage and Mus musculus up to 72 h. The cyanobacterium was identified as Fischerella sp. (accession No. MN593556) through 16S rRNA technique. The compound has potential for use as a therapeutic drug.

Liver X receptors are required for thymic resilience and T cell output.

The thymus is a primary lymphoid organ necessary for optimal T cell development. Here, we show that liver X receptors (LXRs)-a class of nuclear receptors and transcription factors with diverse functions in metabolism and immunity-critically contribute to thymic integrity and function. LXRαβ-deficient mice develop a fatty, rapidly involuting thymus and acquire a shrunken and prematurely immunoinhibitory peripheral T cell repertoire. LXRαβ's functions are cell specific, and the resulting phenotypes are mutually independent. Although thymic macrophages require LXRαβ for cholesterol efflux, thymic epithelial cells (TECs) use LXRαβ for self-renewal and thymocytes for negative selection. Consequently, TEC-derived LXRαβ protects against homeostatic premature involution and orchestrates thymic regeneration following stress, while thymocyte-derived LXRαβ limits cell disposal during negative selection and confers heightened sensitivity to experimental autoimmune encephalomyelitis. These results identify three distinct but complementary mechanisms by which LXRαβ governs T lymphocyte education and illuminate LXRαβ's indispensable roles in adaptive immunity.

Impact of PRRSV strains of different in vivo virulence on the macrophage population of the thymus

The emergence of “highly pathogenic” isolates of porcine reproductive and respiratory syndrome virus (HP-PRRSV) has raised new concerns about PRRS control. Cells from the porcine monocyte-macrophage lineage represent the target for this virus, which replicates mainly in the lung, and especially in HP-PRRSV strains, also in lymphoid organs, such as the thymus. This study aimed at evaluating the impact of two PRRSV strains of different virulence on thymic macrophages as well as after heterologous vaccination. After experimental infection with PR11 and PR40 PRRSV1 subtype 1 strains (low and high virulent, respectively) samples from thymus were analysed by histopathology and immunohistochemistry for PRRSV N protein, TUNEL, CD172a, CD163, CD107a and BA4D5 expression. Mortality was similar in both infected groups, but lung lesions and thymus atrophy were more intense in PR40 group. Animals died at 10–14 dpi after PR11 or PR40 infection showed the most severe histopathological lesions, with a strong inflammatory response of the stroma and extensive cell death phenomena in the cortex. These animals presented an increase in the number of N protein, CD172a, CD163 and BA4D5 positive cells in the stroma and the cortex together with a decrease in the number of CD107a positive cells. Our results highlight the recruitment of macrophages in the thymus, the increase in the expression of CD163 and the regulation of the host cytotoxic activity by macrophages. However, no marked differences were observed between PR11- and PR40-infected animals. Heterologous vaccination restrained virus spread and lesions extent in the thymus of PR40-infected animals.

ULTRAMICROSCOPIC CHANGES OF RATS PARATHYROID GLANDS AND THYMUS AFTER SINGLE ADMINISTRATION OF CYCLOPHOSPHAMIDE AT THE DIFFERENT PERIODS OF OBSERVATION

Introduction: Cyclophosphamide has wide spectrum usage as first-line drug in cancer chemotherapy that is why a detailed study of its effect on individual cell populations is of great interest for science and practice. The interaction of the nervous, immune and endocrine systems plays essential role in the homeostasis maintaining. The aim: This study aimed to investigate the ultramicroscopic changes that occur in the parathyroid glands and thymus of male rats after cyclophosphamide administration. Materials and methods: Twenty-four WAG matured male rats were divided randomly into two groups. The first group served as control and was provided 0.9% soluble sodium chloride. The second group received cyclophosphamide in a dosage 200 mg/kg of body weight of animal by intramuscular single injection. All animals were sacrificed on the 7th and 30th day after injection. Parathyroid gland and thymus specimens were dissected out and processed for electron microscopy. Results: The results showed that cyclophosphamide exposure caused marked ultramicroscopic changes in rats parathyroid glands and thymus. On the 7th day after immunosuppression, the nuclei of parathyrocytes have deep wavy invaginations, amount of the organelles that participate in the protein synthesis is reduced to a minimum in the cytoplasm of the chief cells. Characteristic feature is the appearance of numerous plasma cells and active macrophages in thymus. There is a tendency to normalization of the parathyroid structure on the 30th day after administration of cyclophosphamide and reduction of mitotic activity of lymphocytes in thymus, which points to the development of involution process. Conclusions: This data can be successfully extrapolated from experimental animals to humans.

Adenosine in the Thymus.

Adenosine is an ancient extracellular signaling molecule that regulates various biological functions via activating four G-protein-coupled receptors, A1, A2A, A2B, and A3 adenosine receptors. As such, several studies have highlighted a role for adenosine signaling in affecting the T cell development in the thymus. Recent studies indicate that adenosine is produced in the context of apoptotic thymocyte clearance. This review critically discusses the involvement of adenosine and its receptors in the complex interplay that exists between the developing thymocytes and the thymic macrophages which engulf the apoptotic cells. This crosstalk contributes to the effective and immunologically silent removal of apoptotic thymocytes, as well as affects the TCR-driven T-cell selection processes.

TAK1 regulates resident macrophages by protecting lysosomal integrity

Hematopoietic cell survival and death is critical for development of a functional immune system. Here, we report that a protein kinase, TAK1, is selectively required for resident macrophage integrity during embryogenesis. Hematopoietic lineage-specific deletion of Tak1 gene (Tak1HKO) caused accumulation of cellular debris in the thymus in perinatal mice. Although no overt alteration in thymocytes and blood myeloid populations was observed in Tak1HKO mice, we found that thymic and lung macrophages were diminished. In the in vitro setting, Tak1 deficiency caused profound disruption of lysosomes and killed bone marrow-derived macrophages (BMDMs) without any exogenous stressors. Inhibition of the lysosomal protease, cathepsin B, partially blocked Tak1-deficient BMDM death, suggesting that leakage of the lysosomal contents is in part the cause of cell death. To identify the trigger of this cell death, we examined involvement of TNF and Toll-like receptor pathways. Among them, we found that deletion of Tnfr1 partially rescued cell death. Finally, we show that Tnfr1 deletion partially restored thymic and lung macrophages in vivo. These results suggest that autocrine and potentially paracrine TNF kills Tak1-deficient macrophages during development. Our results reveal that TAK1 signaling maintains proper macrophage populations through protecting lysosomal integrity.

Lactobacillus acidophilus attenuates Aeromonas hydrophila induced cytotoxicity in catla thymus macrophages by modulating oxidative stress and inflammation.

The pathogenesis of Aeromonas hydrophila, a potent fish pathogen, is attributed to its ability to cause motile aeromonad septicaemia leading to apoptosis in a myriad of fish species, including freshwater carp Catla catla. However, the underlying mechanism of antagonistic activity of probiotics against A. hydrophila induced apoptosis is not elucidated due to lack of appropriate in-vitro models. This study reported that the exposure of catla thymus macrophages (CTM) to A. hydrophila markedly induced cellular injuries as evidenced by elevated levels of reactive oxygen species (ROS), reactive nitrogen species (RNS), increased apoptosis, DNA damage and decreased cellular viability. Flow cytometry analysis and Annexin-V/propidium iodide assay further confirmed increased ROS positive cells leading to cell death after infection. The quantitative real-time PCR analysis, also revealed upregulation of inducible nitric-oxide synthase (iNOS), pro-inflammatory cytokine (TNFα), cyclooxygenase2 (COX-2) and downregulation of anti-inflammatory cytokine (IL-10). Pretreatment of cells with probiotic, Lactobacillus acidophilus attenuated A. hydrophila induced apoptosis as evident from the decrease in the levels of ROS, RNS and DNA damage. Significant increase (P≤0.05) in expression of TNFα and IL-10 and decrease in iNOS and COX-2 was observed on probiotic stimulation. In-vivo study using catla fingerlings confirmed similar pattern of ROS, iNOS, NO production and cytokine expression in thymus. This study provides a comprehensive insight into the mechanistic basis of L. acidophilus induced macrophage mediated inflammatory response against A. hydrophila in CTM cells. Further, it speculates the possibility of using cost-effective in-vitro models for screening probiotic candidates of therapeutic potential in aquaculture industry.

Development and characterization of a monoclonal antibody against the putative T cells of Labeo rohita.

In this study, we have described the development and characterization of monoclonal antibodies (MAbs) directed against thymocytes of rohu, Labeo rohita. MAbs were obtained by immunizing BALB/c mice with freshly isolated and nylon wool column enriched mononuclear cells of thymus. Positive clones against thymocytes were screened by cellular ELISA. The hybridoma showing strong reactivity with nylon wool enriched mononuclear cells, and non-reactivity with a rohu thymus macrophage cell line and rohu serum was selected and subjected to single cell cloning by limiting dilution. The MAbs secreted by a positive clone were designated as E6 MAb. Western blotting of reduced protein from enriched thymocytes showed that E6 reacted with a 166.2kDa polypeptide and belongs to the IgG1 subclass. Flow cytometric analysis of gated lymphocytes, revealed that the percentage of E6 positive (E6+) cells in thymus (n=5, 720.4±79.70g) was 89.7%. Similarly, the percentage of E6+ cells in kidney, spleen and blood (n=5) was 6.71, 1.71 and 1.88%, respectively. In indirect immunoperoxidase test, E6+ cells appeared to be lymphoid cells with a high nucleus to cytoplasmic ratio and were densely packed in the central region of thymus whereas, a few cells were found to be positive in kidney and spleen sections. E6 MAb also reacted with a small population of lymphocytes in blood smear. This MAb appears to be a suitable marker for T lymphocytes and can be a valuable tool in studying immune response and ontogeny of L. rohita immune system.

Aberrant tissue positioning of metallophilic macrophages in the thymus of XCL1-deficient mice.

Metallophilic macrophages hold a strategic position within the thymic tissue and play a considerable function in thymic physiology. The development and positioning of these cells within thymic tissue are regulated by complex molecular mechanisms involving different cytokine/chemokine axes. Herein, we studied the role of XCL1 signaling in these processes. We show that in the XCL1-deficient thymus numerous metallophilic macrophages are aberrantly positioned in the thymic cortex, instead of their normal location in the cortico-medullary zone. Still, these cells retain their normal appearance: very large size with prominent, ramifying cytoplasmic prolongations. This shows that XCL1 signaling is not involved in morphological development, but rather in correct positioning of metallophilic macrophages within the thymic tissue. In contrast to thymic metallophilic macrophages, the positioning of splenic marginal metallophilic macrophages is not affected by XCL1-deficiency.

0380: Truncated thioredoxin (Trx-80) promotes pro-inflammatory macrophages of the M1 phenotype and enhances atherosclerosis

Vascular cells are particularly susceptible to oxidative stress that is believed to play a key role in the pathogenesis of cardiovascular disorders. Thioredoxin-1 (Trx-1) is an oxidative stress-limiting protein with anti-inflammatory and anti-apoptotic properties. In contrast, its truncated form (Trx-80) exerts pro-inflammatory effects. Here we analyzed whether Trx-80 might exert atherogenic effects by promoting macrophage differentiation into the M1 pro-inflammatory phenotype. Trx-80 at 1 μg/ml significantly attenuated the polarization of anti-inflammatory M2 macrophages induced by exposure to either IL-4 at 15 ng/ml or IL-4/IL-13 (10 ng/ml each) in vitro, as evidenced by the expression of the characteristic markers, CD206 and IL-10. By contrast, in LPS-challenged macrophages, Trx-80 significantly potentiated the differentiation into inflammatory M1 macrophages as indicated by the expression of the M1 cytokines, TNF-α and MCP-1. When Trx-80 was administered to hyperlipoproteinemic ApoE2.Ki mice at 30 μg/g body weight (b.w.) challenged either with LPS at 30 μg/30 g (b.w.) or IL-4 at 500 ng/30 g (b.w.), it significantly induced the M1 phenotype but inhibited differentiation of M2 macrophages in thymus and liver. When ApoE2.Ki mice were challenged once weekly with LPS for 5 weeks, they showed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. Such effect was potentiated when mice received daily, in addition to LPS, the Trx-80. Moreover, the Trx-80 treatment led to a significantly increased aortic lesion area. The ability of Trx-80 to promote differentiation of macrophages into the classical proinflammatory phenotype may explain its atherogenic effects in cardiovascular diseases.

Development and characterization of a continuous macrophage cell line, LRTM, derived from thymus of Labeo rohita (Hamilton 1822)

A long-term thymic macrophage cell line from the thymus explants of Labeo rohita designated as LRTM (L. rohita thymic macrophages) was established, which has been maintained in culture for more than 1yr. This cell line designated LRTM cells have been subcultured for 70 passages. The cells shape was initially long and elongated; with subsequent passages, the cells became short and epithelial like. The cells exhibited optimum growth in L-15 containing 10% fetal bovine serum and also in Dulbecco's modified Eagle's medium at 37°C with 5% CO2 and showed 85+-% viability after 12mo storage in liquid nitrogen. In addition, cells showed nonspecific esterase and surface expression of Fc receptors for immunoglobulin G and classes I and II major histocompatibility complex antigens. These observations confirmed that this cell line had the morphologic and functional features as a macrophage. The cells exhibited phagocytic activity by engulfing yeast cells as well as fluorescent latex beads, which was demonstrated by scanning electron microscopy and Giemsa staining. The long-term cultured cells show rapid production of reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharides and phorbol miristate acetate (PMA). Mostly, all the cells were alpha napthyl esterase acetate positive. After stimulation with PMA and lipopolysaccharide, cultured fish macrophages produced reactive oxygen and nitrogen intermediates. The karyotype analysis showed that these cells have a tetraploid karyotype with 100 chromosomes in each cell, indicating that they are normal L. rohita cells. Amplification, sequencing, and alignment of fragments of two mitochondrial genes 12S rRNA from rohu confirmed that the cell line originated from L. rohita. This cell line should be useful for studying the role of thymic macrophages in differentiation and maturation of thymocytes and can be source of macrophage-specific enzymes and cytokines. The macrophage cell line will be invaluable in studies of pathogen/macrophage interactions, the mechanisms of macrophage antimicrobial effector functions and the contribution of macrophages to the specific immune responses of teleosts.