Adhesion patterns of cell interactions revealed by reflection contrast microscopy

Reflection contrast in combination with phase contrast microscopy was utilized for the study of adhesion patterns of locomotive L5222 rat leukemia cells. It was found that for cells moving in a spherical shape on the glass surface, adhesions were very faint. This inconspicuous pattern, however, became very distinct, as soon as the cells changed to a flattened configuration. Such a change took place when leukemia cells came into contact with other spread cells and started to move under these cells. Reflection contrast further showed that in the pathway of the locomoting L5222 cells the adhesions of the overlying spread cells were momentarily detached from the substrate. It is concluded that the combination of reflection contrast and phase contrast represents a good tool for gaining new information on the interaction of motility and formation of adhesions.     

Emperipolesis of hematopoietic cells in myelocytic leukemia. Electron microscopic and phase contrast microscopic studies

In case of blast crisis of chronic myelocytic leukemia, the blast cells contained several kinds of normal hematopoietic cells. The peroxidase reaction was strongly positive in the neutrophilic granules of the engulfed neutrophils. These engulfed cells appeared to be normal and the limiting membranes of the engulfing cells seemed to be intact. We speculated therefore that this phenomenon might be emperipolesis. In a case of chronic myelocytic leukemia and a case of acute myelocytic leukemia, some megakaryoblasts showed the same phenomenon. These megakaryoblasts did not phagocytize latex particles. The limiting membranes of the engulfing megakaryoblasts were stained with ruthenium red but those of the engulfed hematopoietic cells were not stained. By phase microscopy, the engulfed cells were actively moving inside the megakaryoblasts and it was observed that the engulfed cells were actually living within the engulfing cells. These results demonstrated that this phenomenon was emperipolesis. Observations with an electron microscope and the phase microscope are indispensable for distinguishing emperipolesis from phagocytosis.     

Fibrillar bodies in leukemic cells revealed by polarization microscopy.

An optical polarizing microscope with a good coefficient of extinction permits the visualization of the cytoplasmic fibrillar body in living preparations and smears of leukemic cells (human leukemias and the L 5222 experimental leukemia). These inclusions are not visible by phase contrast microscopy nor in fixed and stained smears. The detection in living cells of fibrillar bodies makes it possible to study directly the conditions for their formation and their reaction to the effect of certain drugs.     

The role of cellular locomotion in leukemic infiltration. An organ culture study on penetration of L 5222 rat leukemia cells into the chick embryo mesonephros.

The significance of cellular locomotion for leukemic infiltration was investigated using L 5222 rat leukemia cells. Previous cinemicrographic studies have shown that these cells are able to locomote only after formation of a uropod-like posterior extension. This characteristic locomotive configuration of L 5222 cells is easily recognizable in scanning electron micrographs and appropriate sections. Leukemia cells were inoculated on slices of chick embryo mesonephros incubated for 24h; at this time the fragments are completely encapsulated. Leukemic infiltration is found to begin within the first 2 h and to increase gradually up to the end of the observation period at 72 h. Spread of leukemia cells occurs mainly in the intertubular spaces; the tubular epithelium is only rarely affected. In all stages of infiltration, L5222 cells with the characteristic locomotive configuration are frequently recorded. Besides this strong although indirect indication for the significance of locomotion, further evidence was provided by experiments performed at 25 degrees C and 18 degrees C. In accordance with the previous cinemicrographic finding that at these temperatures L 5222 cells are unable to produce their posterior extension, no leukemic infiltration mesonephros fragments is recognizable at subnormal temperatures.     

Augmentation of host antitumor immunity by low doses of cyclophosphamide and mafosfamide in two animal tumor models

Summary By cloning in vitro we have obtained two sublines of the L5222 rat leukemia, one with high (L5222-S) and the other with low (L5222-R) in vivo sensitivities to non-toxic doses of mafosfamide, a stabilized derivative of 4-hydroxy-cyclophosphamide. This sensitivity in vivo was not related to the cytotoxic activity of the drug in vitro. Treatment of rats bearing the L5222-S and of mice transplanted with the MOPC-315 plasmocytoma with low doses of mafosfamide or cyclophosphamide resulted in a high percentage of surviving animals, which were resistant to a subsequent tumor challenge. Viable leukemic cells were needed to establish antitumor immunity, since it was not possible to induce resistance by injection of mitomycin-C-treated, non-viable L5222 cells. The adoptive transfer of spleen cells from animals immune against the L5222-S and the MOPC-315 resulted in resistance of the syngeneic recipients against a rechallenge with tumor cells, provided that the animals were treated with an immunosuppressive dose (100 mg/kg) of cyclophosphamide prior to the spleen cell implantation. In nude mice treatment of the L5222 with low doses of mafosfamide also resulted in surviving animals, however resistance to a second tumor challenge occurred only sporadically.The data presented confirm that therapy with cyclophosphamide or mafosfamide enhances host antitumor immunity but, contrary to previous reports, it could be demonstrated that successful tumor rejection was independent of T cells.Supported by the Federal Ministry of Research and Technology (BMFT), Bonn-Bad Godesberg, FRG     

Different modes of mesenteric infiltration displayed by two rat leukemias. A study by scanning and transmission electron microscopy and by microcinematography

Infiltration of the mesentery after intraperitoneal implantation of two transplantable rat leukemias, the undifferentiated L5222 and the myeloid BNML, was studied by means of scanning and transmission electron microscopy, and microcinematography. In animals implanted with L5222 cells, contraction of the mesenteric mesothelium is a conspicuous feature. It occurs within the first 24h after implantation and influences decisively the course of infiltration. In contrast, The presence of BNML cells leads to mesothelial contraction only in the terminal stage and, therefore, exerts no direct effect on infiltration. In addition, the two leukemias differ with regard to their cellular motility. Whereas L5222 cells locomote within the mesentery, only stationary movements are recorded with BNML cells. Based on the different interactions with the mesothelium and cell motilities, two distinct modes of infiltrating the mesentery could be ascertained for the two rat leukemias.     

Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.     

Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.     

Ultrastructural evidence for contacts between migrating L5222 rat leukemia cells and extracellular matrix components of the rat mesentery

The nature of interactions between cells migrating through tissues and their structural surroundings are largely unknown. We have therefore examined the ultrastructural relationship between L5222 rat leukemia cells, moving through the loose connective tissue of the mesentery, and components of the extracellular matrix (ECM). Ultrathin tissue sections, fixed in the presence of ruthenium hexammine trichloride (RHT), revealed the following: Constitutents of fibrillar and nonfibrillar elements of the ECM are in contact with the plasma membrane of L5222 cells. Linear nonfibrillar ECM elements contact the plasma membrane at point-like sites, often associated with root-like structures present within the submembraneous microfilament mesh. Aggregates of ECM material are connected to patch-like cell membrane sites, associated with a condensed, plate-like part of the microfilament mesh. Point-like and patch-like contacts are more numerous at the anterior part of polarized migrating L5222 cells than on the posterior end. In round resting leukemia cells they are evenly distributed around the cell periphery. We suggest that the ECM-cell membrane contacts represent tissue adhesion sites. We therefore hypothesize that in migrating cells a coordinate interaction occurs between the contact sites and the continuous microfilament meshwork which results in a simultaneous backward movement of ECM-membrane contacts on the cell body and in a net forward movement of the whole cell. Since Dembo et al. (1981) present a similar mechanism for in vitro locomotion of granulocytes, we assume that blood cell locomotion in vivo and in vitro depends on similar molecular mechanisms: force generation by the cell, transmembraneous linkage between cytoskeletal and ECM elements, and membrane fluidity. The major difference in blood cell locomotion through a three-dimensional tissue or on a plane substratum would then be given by the distribution of contact sites, occurring around the cell periphery or limited to the ventral cell surface, respectively.     

Lack of mast cell reactivity during leukemic infiltration of the rat mesentery under syngeneic and allogeneic conditions. A study by transmission electron microscopy (TEM)

The ultrastructural morphology of mast cells localized in rat mesenteries was studied after intraperitoneal implantation of L5222 rat leukemia cells in syngeneic and allogenic hosts. It became evident that the mast cells in the syngeneic (BDIX rat) as well as the allogeneic system (BN rat) showed nor morphological alterations. Degranulation was never observed. This is in contrast to the behavior of macrophages which displayed a strong phagocytotic activity in allogeneic hosts. Thus, it seems that mast cells, under the present experimental conditions, remained inactive during a phase of intense tumor rejection.     

Comparative morphology of cells located on glass and in the loose connective tissue: a study by scanning electron microscopy

Most of the studies dealing with cellular shape, surface configuration, and motility are carried out in vitro on plane substrata. During the past years, the direct transfer of results obtained under these conditions to the cellular behavior displayed in the living organism, has been increasingly challenged. For this reason we have investigated the above mentioned functions of different cell classes localized on glass and in the loose connective tissue. The cells utilized were: fibroblasts and macrophages from normal rat and rabbit mesenteries, V2 rabbit carcinoma cells and L5222 rat leukemia cells. The combination of time-lapse cinematography and scanning electron microscopy revealed that motility and surface features are the same, irrespective of the immediate surrounding. Cellular shape and attachment, on the other hand, are dependent on the substrate. Fibroblasts, macrophages and cells of epithelial origin, including carcinoma cells, flatten on glass, but have a rounded configuration in the tissue. The flat leading lamellae displayed during locomotion on glass, are not evident in cells migrating through tissues. What regards attachment devices, extensively studied on glass, their formation and position within a tissue are, at present, a matter of speculation. Although it can be assumed that a similar process is operable in vivo and in vitro, clarification rests upon the use of ultrahistochemical techniques.     

Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission

The spread of retroviruses between cells is estimated to be 2-3 orders of magnitude more efficient when cells can physically interact with each other. The underlying mechanism is largely unknown, but transfer is believed to occur through large-surface interfaces, called virological or infectious synapses. Here, we report the direct visualization of cell-to-cell transmission of retroviruses in living cells. Our results reveal a mechanism of virus transport from infected to non-infected cells, involving thin filopodial bridges. These filopodia originate from non-infected cells and interact, through their tips, with infected cells. A strong association of the viral envelope glycoprotein (Env) in an infected cell with the receptor molecules in a target cell generates a stable bridge. Viruses then move along the outer surface of the filopodial bridge toward the target cell. Our data suggest that retroviruses spread by exploiting an inherent ability of filopodia to transport ligands from cell to cell.     

Modulation of natural cytotoxicity by alloantibodies. I. Alloantisera enhancement of cytotoxicity of mouse spleen cells toward a human myeloid cell line.

Natural killer activity of spleen cells obtained from different strains of mice against the human myeloid leukemia cell line, K562, and two mouse cell lines P815 and L1210 was measured by using the 4-hr chromium release assay. The level of cytotoxic activity of spleen cells against the K562 target was usually less than 4% lysis. However, treatment of the spleen cells with a specific anti-H-2 antiserum resulted in a dose-dependent augmentation of the degree of lysis of K562 cells. The augmentation of cytotoxic activity could be obtained by pretreatment of the spleen cells with antisera or by directly adding the antisera to the cytotox-incubation medium. Anti-thy-1 and anti-immunoglobulin antisera had no enhancing effect under similar conditions. The specific alloantisera-treated spleen cells did not show any increase in cytotoxicity against P815 and L1210 target cells. Spleen cells responsible for the alloantiserum-mediated augmentation of cytotoxicity against K562 cells appear to be different from T or B cells as indicated by their resistance to anti-thy-1 and complement treatment and lack of adherence to nylon wool columns.     

Avoiding the void: cell-to-cell spread of human viruses

The initial stages of animal virus infection are generally described as the binding of free virions to permissive target cells followed by entry and replication. Although this route of infection is undoubtedly important, many viruses that are pathogenic for humans, including HIV-1, herpes simplex virus and measles, can also move between cells without diffusing through the extracellular environment. Cell-to-cell spread not only facilitates rapid viral dissemination, but may also promote immune evasion and influence disease. This Review discusses the various mechanisms by which viruses move directly between cells and the implications of this for viral dissemination and pathogenesis.     

A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin.

The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell-to-cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium-host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.     

Emperipolesis as a key feature in imprint cytology of the thymus. A report of two cases

BACKGROUND: Imprint cytology of the thymus has not received much attention. Cytology of the thymus is important because the uninvolved thymus may be needled during aspiration procedures. CASES: In two cases, during surgery for carcinoma of the thyroid, we received thymic tissue mistakenly sampled as a pretracheal lymph node for frozen section to rule out metastasis. Imprint smears were studied. The presence of thymocytes in the cytoplasm of thymic epithelial cells (emperipolesis) was the most significant feature in the imprints. However, it was not detected on histology. CONCLUSION: Thymic epithelial cells provide mechanical support and play a major role in the maturation of lymphocytes (thymocytes). They are observed as emperipolesis on imprint cytology. Its utility in identifying thymic cells in aspiration cytology needs to be investigated.     

Attenuation of CXCR4 responses by CCL18 in acute lymphocytic leukemia B cells

CCL18 and CXCL12 are homeostatic chemokines with high constitutive concentrations in serum. Elevated levels of CCL18 have been described in various diseases including childhood acute lymphocytic leukemia (ALL) but its functions remain poorly characterized. Its receptor has not been identified, but functional cellular responses like lymphocyte chemotaxis have been described. CXCL12 is a pivotal chemokine for hematopoiesis and B cell homing processes. We demonstrate that CCL18 interferes with CXCL12‐mediated pre‐B ALL cell activation. CXCL12‐induced calcium mobilization, chemotaxis, pseudo‐emperipolesis and cellular proliferation could be significantly reduced by CCL18 in pre‐B ALL cell lines. The results could be observed in primary cells from patients suffering from pre‐B ALL, but not in cells from patients suffering from common ALL. Direct effects of CCL18 on the receptor for CXCL12, CXCR4, could be excluded. Moreover, we found that CCL18 modulations of CXCL12‐induced responses are mediated through the chemokine‐like receptor GPR30. CCL18 bound to GPR30 expressing cells, and antibodies against GPR30 abolished this binding as well as CCL18‐mediated functional effects. We also observed that, CCL18 interferes with the activation of GPR30 by previously identified ligands (17β‐estradiol and chemical agonists). We therefore suggest that CCL18 is an important modulator of CXCR4‐dependent responses in pre‐B ALL cells via interactions with GPR30. J. Cell. Physiol. 225: 792–800, 2010. © 2010 Wiley‐Liss, Inc.     

Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages

Leptospira interrogans is a spirochaete responsible for a zoonotic disease known as leptospirosis. Leptospires are able to penetrate the abraded skin and mucous membranes and rapidly disseminate to target organs such as the liver, lungs and kidneys. How this pathogen escape from innate immune cells and spread to target organs remains poorly understood. In this paper, the intracellular trafficking undertaken by non-pathogenic Leptospira biflexa and pathogenic L. interrogans in mouse bone marrow-derived macrophages was compared. The delayed in the clearance of L. interrogans was observed. Furthermore, the acquisition of lysosomal markers by L. interrogans-containing phagosomes lagged behind that of L. biflexa-containing phagosomes, and although bone marrow-derived macrophages could degrade L. biflexa as well as L. interrogans, a population of L. interrogans was able to survive and replicate. Intact leptospires were found within vacuoles at 24 h post infection, suggesting that bacterial replication occurs within a membrane-bound compartment. In contrast, L. biflexa were completely degraded at 24 h post infection. Furthermore, L. interrogans but not L. biflexa, were released to the extracellular milieu. These results suggest that pathogenic leptospires are able to survive, replicate and exit from mouse macrophages, enabling their eventual spread to target organs.     

Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20–243 CrossMab

Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an “Achilles heel” for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20–243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20–243 CrossMab would be a promising therapeutic agent against lymphoma.     

Emperipolesis in a common breast malignancy. A case report

BACKGROUND: Emperipolesis is a phenomenon characterized by the presence of leukocytes/lymphocytes within the cytoplasm of other cells. The present report describes this unusual observation within epithelial cancer cells of the breast. CASE: A 52-year-old female presented with a hard, adherent lump over the right breast for one year. Fine needle aspiration and histopathologic examination of the tumor showed features of infiltrating duct carcinoma with emperipolesis as a striking feature of the tumor cells. The tumor showed a near-total response to neoadjuvant chemotherapy. CONCLUSION: The mechanism and biologic significance of emperipolesis in producing a near-total response to neoadjuvant chemotherapy in the present case suggest its role in inducing a tumoricidal effect, possibly involving a cascade of chemokines.