Morphological differences between sub-populations of human lymphocytes revealed by scanning electron microscopy.

Human lymphoid cells were examined by scanning electron microscopy (SEM) to see if a correlation existed between surface morphologic features and the presence of various surface markers and receptors. When viewed by SEM thymocytes appeared as smooth-surfaced cells with few surface microvilli; peripheral blood lymphocytes (PBL) on the other hand were moderately to densely villate with no entirely smooth-surfaced cells observed. Surface morphology within PBL samples was not uniform, due mainly to variations in the shape and number of microvilli. However, 2 distinctive types of surface morphology (termed Types 1 and 2) were discernable with a small number of cells displaying features of both groups (Type 3). The majority of E-rosette forming cells (T lymphocytes) displayed Type 1 and the majority of cells bearing demonstrable surface immunoglobulin (B lymphocytes) displayed Type 2 morphology. Exposure of PBL to anti-T cell specific ALG resulted in cytolysis of cells with Type 1 morphology while cells with Type 2 morphology appeared largely unaffected. PBL with Fc and C3 receptors displayed all 3 types of morphology. It is concluded that T and B lymphocytes do have subtle but nevertheless discernable differences in surface morphology and within these 2 groups, variations in surface morphology are probably associated with changes in the physiological status of the cell.     

Mitogen stimulation of chronic lymphocytic leukemic lymphocytes: defective phytohemagglutinin stimulation independent of immunologic cell surface markers

Peripheral blood lymphocytes (PBL) from 107 untreated patients with chronic lymphocytic leukemia (CLL) were analyzed for the presence of surface immunoglobulin (Ig) and the ability to form rosettes with sheep erythrocytes (SRBC). Four groups were identified based on the cell surface markers: (1) 81 patients' PBL expressed primarily IgM kappa or IgM lambda, 4 further patients' PBL expressed IgM with equal percentages of kappa and lambda surface markers; (2) 13 patients had equal percentages of PBL expressing lg and SRBC receptors; (3) 6 patients' PBL primarily formed rosettes with SRBCs, and (4) in 3 patients and the majority of cells had no detectable markers (null cells). Lymphocytes from all patients within each group were tested for their ability to respond to phytohemagglutinin (PHA) and pokeweed mitogen (PWM). The maximum response in PHA-stimulated normal cell cultures appeared at 2--3 days; for PWM-stimulated cultures, maximal response was at 3--5 days. CLL cultures from all patients in each of the four groups required 5--7 days to develop a maximal PHA response. The response of CLL lymphocytes in all groups to PWM stimulation was similar to normal lymphocytes. Thus, the abnormal PHA response of CLL lymphocytes was independent of the presence or pattern of cell surface markers.     

Prolymphocytic leukaemia: surface morphology in 21 cases as seen by scanning electron microscopy and comparison with B-type CLL and CLL in ''prolymphocytoid'' transformation

The surface architecture of leukaemic cells obtained from 21 cases of proven prolymphocytic leukaemia (PLL) and eight cases of chronic lymphocytic leukaemia (CLL) with 'prolymphocytoid' transformation (PL-CLL) was compared with the cell surface morphology of leukaemic cells obtained from 46 cases of B-type CLL, using the scanning electron microscope (SEM). All cases were defined by cytochemistry, immunological markers and transmission electron microscopy prior to SEM examination. B-CLL cells showed the well-recognized spectrum of surface architecture described in earlier studies. The majority of cells had moderate numbers of short microvilli, although in a minority, cells with relatively smooth surfaces predominated. In seven of the eight cases of PL-CLL, cells were villous in nature and in this respect similar to CLL cells; however, more cells with dense microvilli were seen. The prolymphocytic cells were recognized by their larger size and in 18 of the 19 cases of B-derived PLL, villous cells predominated. Two cases of T-derived PLL showed variable cell surface morphology ranging from smooth to moderately villous. It appears that B-PLL cells are most frequently villous and display more surface microvilli than B-CLL cells. B-prolymphocytes display the surface features regarded as characteristic for neoplastic B-cells as seen in patients with B-type lymphoma and leukaemia.     

Surface Features of Sezary Cells: A Scanning Electron Microscopy Study of 5 Cases

The surface features of circulating cells from 5 patients with typical Sezary's Syndrome (SS) are described using scanning electron microscopy (SEM). Sezary cells prepared by different methods, with and without prior fixation in cell suspension, showed similar surface architectures. SS cells were mostly spherical and moderate to markedly villous in appearance, and in this respect, resembled the majority of circulating lymphocytes from patients with chronic lymphocytic leukaemia (CLL). A proportion of cells were larger and more irregular in shape while others had small extensions of cytoplasm resembling small uropods with clusters of polarised microvilli. Despite the latter findings, most SS cells cannot be distinguished from CLL cells on the basis of their surface architecture under the SEM.     

Surface features of Sezary cells: A scanning electron microscopy study of 5 cases.

The surface features of circulating cells from 5 patients with typical Sezary's Syndrome (SS) are described using scanning electron microscopy (SEM). Sezary cells prepared by different methods, with and without prior fixation in cell suspension, showed similar surface architectures. SS cells were mostly spherical and moderate to markedly villous in appearance, and in this respect, resembled the majority of circulating lymphocytes from patients with chronic lymphocytic leukaemia (CLL). A proportion of cells were larger and more irregular in shape while others had small extensions of cytoplasm resembling small uropods with clusters of polarised microvilli. Despite the latter findings, most SS cells cannot be distinguished from CLL cells on the basis of their surface architecture under the SEM.     

Morphological abnormalities in the lymphocytes of patients with the Wiskott-Aldrich syndrome

Lymphocytes from 18 patients with the Wiskott-Aldrich Syndrome (WAS) were examined by scanning electron microscopy (SEM). Most peripheral blood lymphocytes from normal individuals are covered with slender microvillus projections, but a large proportion of lymphocytes from WAS patients were found to be relatively devoid of microvilli. A lymphocyte morphology scoring system was developed to quantify the density of microvilli: Grade 4 classified those lymphocytes with greater than 75% of the surface covered with microvilli with progressive decrements to grade 1, which were those without microvilli. The mean lymphocyte morphology score of eight normal individuals was 3.62 +/- .22. The mean lymphocyte score of WAS patients was substantially lower (2.89 +/- .27, P less than .001). In addition, WAS lymphocytes often were qualitatively abnormal, with short, blunted microvilli. These morphological criteria were used to diagnose WAS from the cord blood lymphocytes of one "at-risk" patient. Thus, WAS is the first primary immunodeficiency in which morphological abnormalities have been identified that can aid in diagnosis.     

Characterization of a suppressor T-cell chronic lymphocytic leukemia with ADCC but not NK activity

A patient with T-cell chronic lymphocytic leukemia (T-CLL) is reported whose cells demonstrate in vitro suppression of normal lymphocyte mitogen stimulation. The patient, who remains in Rai's clinical stage 0 on no therapy after more than 24 mo of observation, has shown a less aggressive clinical course than is usually attributed to T-CLL. His peripheral blood lymphocytes (PBL) were characterized by functional assays as well as surface markers. Over 90% of the patient's PBL formed rosettes with sheep erythrocytes and were lysed by two T-cell-specific antisera plus complement, while less than 1% bore surface immunoglobulins, and only 3% had complement receptors. In addition, 45% of the PBL demonstrated Ia-like antigens, more than 50% expressed a receptor for the Fc portion of IgG(T gamma), and most of the sheep erythrocyte rosettes were inhibited by theophylline. The patient's cells failed to respond to several mitogens and they caused marked suppression of lymphoproliferative responses to normal PBL to phytohemagglutinin (PHA) and concanavalin A (Con-A). The patient's lymphocytes also exhibited antibody-dependent cytotoxic activity (ADCC) against antibody-coated nucleated target cells, but lacked demonstrable natural killer (NK) activity. This patient's T-CLL cells appear to represent the clonal expansion of a subset of T cells with a previously undescribed pattern of suppressor and cytotoxic activities.     

Scanning electron microscopic study of leukemic human B lymphocytes.

Peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (CLL), lymphoplasmacytoid lymphoma, centrocytic lymphoma and hairy cell leukemia were studied by scanning electron microscopy (SEM). In general, SEM revealed rather homogenous cell populations. Most lymphocytes displayed a moderately villous surface architecture, although smooth surfaces predominated in 3 cases with CLL and in 1 case with lymphoplasmacytoid lymphoma. Hairy cells showed surface features of both lymphocytes and monocytes. The results indicate that leukemic B and T lymphocytes cannot be distinguished by SEM alone.     

Expression of Th1 markers by lung accumulated T cells in pulmonary sarcoidosis

OBJECTIVES: The balance between Th1 and Th2 T cells, classified by virtue of their cytokine production can in an immune response influence the phenotype and progression of several clinical diseases. In this study, we examined the expression of Th1 associated chemokine and cytokine receptors CXCR3, CCR5, and interleukin (IL)-12R, IL-18R, respectively, as well as of the Th2 associated chemokine receptors CCR4 and CXCR4 on CD4+ and CD8+ T cells. SUBJECTS: Eighteen patients with untreated pulmonary sarcoidosis. MATERIALS AND METHODS: We used monoclonal antibodies and flow cytometry to analyse the expression of chemokine receptors CXCR3, CXCR4, CCR4 CCR5 and cytokine receptors IL-12R, IL-18R in combination with anti-CD4 and anti-CD8 mAbs in bronchoalveolar lavage fluid (BAL) and peripheral blood lymphocytes (PBL) from sarcoidosis patients. RESULTS: There were significantly more BAL CD4+ T cells expressing CXCR3, CCR5, IL-12R and IL-18R compared with paired PBL CD4+ T cells. In contrast, the Th2 associated chemokine receptors CXCR4 and CCR4 were expressed by a fewer percentage of BAL CD4+ compared with PBL CD4+ T cells. There was a positive correlation between the percentage of BAL lymphocytes and the number of CXCR3 and CCR5 expressing CD4+ BAL T cells. Also, the number of CD4+ IL-18R+ BAL fluid cells correlated negatively with disease duration. CONCLUSIONS: The lung accumulation of CXCR3, CCR5, IL-12R and IL-18R expressing T cells is in line with previous reports showing elevated levels in the lung of the corresponding ligands in sarcodosis. Blocking such ligands and/or receptors may develop into a future immunomodulatory therapy.     

Temperature-Induced Variations in the Surface Topology of Cultured Lymphocytes Are Revealed by Scanning Electron Microscopy

Temperature-induced variations in the surface morphology of cultured lymphocytes were evaluated by scanning electron microscopy. At 25-37 degrees the cells' surfaces are largely obscured by numerous undulating microvilli of various lengths but uniform diameter. Temperature changes alter the number of microvilli, their lengths, diameters, distribution, branching, and fusing. Typically, chilling to 0-4 degrees markedly reduces the number of microvilli and increases the diameter of the survivors in a reversible process. In contrast, heating the cells to about 45 degrees rapidly and irreversibly transforms the ordinarily smooth membrane surface into one with a "cobblestone" morphology. At the same time most microvilli disappear and the few that remain clump into a cap. The data suggest that the low-temperature effects reflect a change in the physical state of membrane lipids, while the high-temperature alterations represent thermotropic protein transitions.     

Surface Immunoglobulin of Human Lymphocytes

Using ferritin as surface marker, the localization of the surface immunoglobulin (Ig) was studied on peripheral lymphocytes from normal human individuals and patients with macroglobulinaemia Waldenström by scanning immunoelectron microscopy. Normal IgG-, IgM-lymphocytes and pathological IgM-lymphocytes were then compared with regard to their topographical differences. In all cells examined, IgG- and IgM-conjugated ferritin particles were detected all over the cell surface, but the distribution of the former on the normal IgG-lymphocytes was slightly more diffuse than that of the latter on the normal and pathological IgM-lymphocytes. Furthermore, in the pathological IgM-lymphocytes, the clustered IgM-conjugated ferritin particles were found in great number on the microvilli. Normal IgG-lymphocytes were almost always characterized by short rod-like microvilli standing densely and vertically on the cell surface. Some of normal IgM-lymphocytes had a similar appearance to those of normal IgG-lymphocytes (type A) but others (type B) had tilted rod-like microvilli or wide plate-like processes on their surface. As for IgM-lymphocytes of macroglobulinaemia, most lymphocytes had tilted rod-like microvilli and wide plate-like processes similar to type B, whereas a minor population of the pathological lymphocytes carried long, thin rod-like microvilli standing vertically on the surface.     

Heterogeneity of immunologic markers and surface morphology in childhood lymphoblastic lymphoma

The neoplastic cells from seven patients with childhood lymphoblastic lymphoma were studied for cell surface markers and surface morphology in the scanning electron microscope (SEM). The cells were studied for surface immunoglobulin (Slg), complement receptors (EAC), receptors for cytophilic antibody (IgGEA), and nonimmune rosette formation with sheep red blood cells (E). In one patient the cells exclusively bound E, suggesting a T-lymphocytic origin. In two patients the cells bound EAC, but demonstrated no other B-lymphocytic markers. In two patients no markers were detected, and in two patients receptors for both E and EAC were demonstrated. Additional studies in one of these patients permitted simultaneous demonstration of both markers on the same neoplastic cells. The neoplastic cells were also examined by SEM after fixation and critical point dehydration. No consistent surface morphology was observed. In four patients the cells were predominately smooth, whereas in two patients variable numbers of surface microvilli were present. A correlation of the surface features with membrane markers could not be established. A comparison of the surface markers with clinical and cytologic features revealed clinical homogeneity in spite of the heterogeneous immunologic markers. This heterogeneity was most likely a reflection of neoplastic alteration and disordered differentiation of the cells. The observation of complement receptors on the cells of four cases is a feature not previously reported in this disease and should be investigated in other presumed T-cell malignancies.     

Ligand-independent cap formation: redistribution of surface receptors on mouse lymphocytes and thymocytes in hypertonic medium.

Most of the mobile receptors on mouse lymphocytes and thymocytes, including immunoglobulins, H-2 antigens, Thy-1.2 antigens, some concanavalin A receptors, and some antigenic determinants detected by anti-thymocyte serum, were redistributed into caps when the cells were incubated in hypertonic medium (about 600 mOsM) in the absence of ligands. The caps reverted to the original distributions if the cells were transferred again to isotonic medium. The viability of the cells was not decreased after incubation in the hypertonic medium. Ligand-independent cap formation appeared to depend upon cellular metabolism. Different species of receptors appeared to move with different mobilities during the process of ligand-independent cap formation. Most microvilli on cells showing caps in hypertonic medium were associated with the regions of the caps. These results suggest that free receptors can be induced to form caps if the receptors are allowed to interact with the machinery of cap formation under special conditions.     

Acute lymphoblastic leukemia: A study of 25 cases by scanning electron microscopy

Summary Cells from 25 cases of acute lymphoblastic leukemia (ALL) were studied under the scanning electron microscope (SEM). In 24 of the cases, the vast majority of circulating leukaemic cells had few microvilli. Villous cells were rarely encountered and prominent ridge-like profiles and ruffled membranes were not seen. Only six cases were studied by immunological techniques and four of the cases were of the null type while in two the cells bore detectable T-markers. It seems that ALL is almost always associated with the presence of cells with few microvilli in the peripheral circulation, differing in this respect from most cases of CLL. Although circulating leukaemic lymphocytes with few microvilli are sometimes seen in CLL, the most frequent cell type encountered is a more villous lymphocyte.Differences between leukaemic cells from patients with ALL, CLL and non-lymphoblastic leukaemias are discussed. It appears that SEM may help to distinguish lymphoblastic and nonlymphoblastic leukaemic cells in many instances and can be used as a useful adjunct to other modes of microscopy in the diagnosis of acute leukaemia.

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Zusammenfassung Bei 25 Patienten mit akuter Lymphoblasten-Leukämie (ALL) wurden die Leukämiezellen mit Hilfe des Raster-Elektronenmikroskops untersucht. Bei 24 Patienten fanden sich überwiegend glatte Zellen mit nur wenigen Mikrovilli. Bei diesen Patienten wurden nur wenige zottige Zellen gefunden; Zellen mit prominenten Streifenprofilen und gefalteten Membranen wurden nicht beobachtet. 6 Fälle wurden zusätzlich mit immunologischen Techniken untersucht; in 2 Fällen waren T-Zell-Marker nachzuweisen, 4 gehörten zur sogenannten Null-Fraktion. Die ALL zeigt also fast immer glatte Zellen, im Gegensatz zu den meisten Fällen von chronisch-lymphatischer Leukämie (CLL). Bei der letzteren sind zwar gelegentlich auch glatte Zellen zu sehen; häufigster Zelltyp ist jedoch eine stärker mit Zotten versehene Zelle.Die Unterschiede der Zelloberfläche bei CLL, ALL und anderen Leukämien werden besprochen. Die Raster-Elektronenmikroskopie erlaubt in vielen Fällen eine Unterscheidung zwischen lymphatischen und nicht-lymphatischen Zellen und ist eine wertvolle Ergätnzung zu anderen mikroskopischen Methoden bei der Leukämie-Diagnose.

     

Surface Features of Human Eosinophils: a Scanning and Transmission Electron Microscopic Study of a Case of Eosinophilia

Cells were obtained from the peripheral blood of a patient with marked non-leukaemic eosinophilia. Transmission electron microscopy (TEM) showed typical mature eosinophils. The surface architecture of eosinophils by scanning electron microscopy (SEM) shows that most were spherical with varying numbers of microvilli; a smaller proportion of cells had ridge-like profiles, small ruffles and occasionally blebs. The surface features of the eosinophil thus resemble most lymphocytes and this finding must be considered when leucocyte populations are examined by SEM.     

The Sézary syndrome cell: surface ultrastructural characteristics.

Peripheral blood mononuclear cells from a patient with Sézary syndrome which lacked E-rosette-forming ability and surface immunoglobulins, and which displayed a markedly depressed response to a variety of mitogens, were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) on 3 occasions. The first peripheral blood sample (smooth) differed significantly from two later samples (moderate numbers of microvilli) when surface characteristics were examined by SEM; these differences were confirmed by TEM. The Sézary syndrome cells in this patient may be related to a T lymphocyte which has lost certain surface markers and mitogen response characteristics through a process of de-differentiation.     

High multidrug resistance (P-glycoprotein 170) expression in inflammatory bowel disease patients who fail medical therapy

BACKGROUND & AIMS: The multidrug resistance (MDR) gene codes for a drug efflux pump P-glycoprotein 170 (Pgp-170) expressed on the surface of lymphocytes and intestinal epithelial cells. Inflammatory bowel disease (IBD) poorly responsive to medical therapy may relate to MDR expression because glucocorticoids are known Pgp-170 substrates. METHODS: Using flow cytometry, we measured peripheral blood lymphocyte (PBL) MDR in 153 IBD patients and 50 healthy volunteers, and assessed the relationship between PBL, mucosal intraepithelial lymphocyte (IEL), and mucosal epithelial cell (EC) MDR expression in a further 20 IBD patients and 19 controls. RESULTS: Compared with controls, PBL MDR was significantly elevated in patients with Crohn's disease who required bowel resection for failed medical therapy (mean +/- SEM, 26.7 +/- 2.8 vs. 11.9 +/- 1.0; P <0.0001) and patients with ulcerative colitis who required proctocolectomy for failed medical therapy (20.3 +/- 2.5 vs. 11.9 +/- 1.0; P = 0.001). PBL MDR remained stable over time and was not influenced by disease activity or glucocorticoid therapy. Both PBL and mucosal MDR expression appeared independent of disease activity, and there was a significant correlation between PBL MDR expression and both IEL expression (r = 0.92; P < 0.0001) and EC expression (r = 0.54; P < 0.001). CONCLUSIONS: PBL and mucosal MDR expression may play an important role in determining the response of IBD patients to glucocorticoid therapy.     

Phenotypic analysis of lamina propria lymphocytes. Predominance of helper-inducer and cytolytic T-cell phenotypes and deficiency of suppressor-inducer phenotypes in Crohn's disease and control patients

To better define the nature of intestinal T cells, the phenotypes of isolated lamina propria lymphocytes (LPL) were determined in both Crohn's disease patients and control patients using combinations of monoclonal antibodies that have been found to correlate with particular immunoregulatory functions. Isolated LPL and autologous peripheral blood lymphocytes (PBL) were stained with multiple combinations of monoclonal antibodies and studied by dual immunofluorescence flow cytometry. In LPL, compared with PBL, there was a significant increase in the proportion of T cells having the Leu-3+, Leu-8- and Leu-3+, 2H4- phenotypes (associated with helper-inducer function) and a corresponding decrease in the proportion of T cells having the Leu-3+, Leu-8+ and Leu-3+, 2H4+ phenotypes (associated with suppressor-inducer function). It was also found that in LPL, compared with PBL, the percentage of cells with the Leu-2+, Leu-15+ phenotype (associated with suppressor-effector function) was significantly lower. However, the percentage of T cells with the Leu-2+, 9.3+ phenotype (associated with cytolytic function) was similar in PBL and LPL in control patients. There were no major differences comparing Crohn's disease patients with control patients, except that the proportion of Leu-2+, 9.3+ lymphocytes was higher in PBL in Crohn's disease patients. These results show that the lymphocyte subpopulations in the lamina propria differ from those in peripheral blood in having predominantly the phenotypes of helper-inducer and cytolytic T cells, whereas the phenotypes of suppressor-inducer cells and activated suppressor cells are less frequently observed.     

Reduced expression of chemokine receptors on peripheral blood lymphocytes in patients with hepatocellular carcinoma

OBJECTIVES: Hepatocellular carcinoma (HCC) is a rapidly progressive malignancy. Chemokine receptors are important mediators of lymphocyte migration in cancer. This study evaluated expression of chemokine receptors on lymphocytes of HCC patients. METHODS: Chemokine receptor expression on peripheral blood lymphocytes (PBL) was determined by flow cytometry and RT-PCR. Tumor infiltrating lymphocytes (TIL) and adjacent nontumor liver infiltrating lymphocytes (NIL) were also studied. RESULTS: The expressions of CCR5, CCR6, and CXCR3 on PBL were significantly reduced in HCC patients compared with normal controls, which occurred concurrently with increased expression of the chemokine receptors in TIL and NIL. Reduced expression of CXCR3 on PBL correlated with large tumor size and advanced tumor stage. The reduced chemokine receptor expression was consistent with the reduced mRNA levels and intracellular protein levels in PBL. HCC patients exhibited lower proportions of CD4(+) and CD8(+) T cells with CCR5, CCR6, and CXCR3 expression on PBL, which occurred concurrently with the increased expression of these chemokine receptors on TIL and NIL. The reduced CCR6 and CXCR3 expression on PBL correlated with the reduced memory phenotype in circulation and increased memory phenotype in liver. Furthermore, CCR5-expressing memory T cells were increased in liver compartment compared with circulation. CONCLUSION: This study demonstrated that reduced chemokine receptor expression on PBL was concurrent with increased chemokine receptor expression on both TIL and NIL in HCC. The results demonstrated the role of chemokine receptors in recruitment of lymphocytes from peripheral blood to HCC. The findings have important implications in understanding of immunopathogenesis of HCC.     

Human immunodeficiency virus infection of megakaryocytic cells

The human immunodeficiency virus (HIV) is capable of infecting certain cells of hematopoietic lineage, particularly monocyte-macrophages and T lymphocytes. Recently, the possibility that cells of megakaryocytic lineage are susceptible to HIV infection has been raised. We have characterized infection of the permanent megakaryocytic cell line CMK by HIV in vitro. CMK cells were easily infected by HIV type 2 (HIV-2), producing significant amounts of virus in culture. Infection appeared to be mediated by the CD4 surface antigen on CMK cells. Three different strains of HIV-1 were able to minimally infect CMK cells, suggesting there may be isolates of HIV tropic for megakaryocytes. Infection of CMK cells led to downregulation of the CD4 surface antigen but no discernable change in expression of megakaryocyte-associated proteins glycoprotein Ib and glycoprotein IIb/IIIa. These observations support the likelihood that megakaryocytes are susceptible to HIV infection, and cell lines of megakaryocytic origin may provide a useful model to study effects of the retrovirus on megakaryocyte function.