d-Penicillamine in Vivo Enhances Lymphocyte DN A Synthesis: Role of Macrophages

Administration of d -penicillamine (50 mg/kg/day orally) for 4 days increased the uptake of ³H-thymidine (³H-TdR) in unstimulated and concanavalin -A-stimulated unseparated lymph node and spleen cells from Lewis rats. Increased ³H-TdR incorporation was also found in cultures depleted of adherent cells. d -Penicillamine treatment did not increase the incorporation of ³H-TdR in lymph node and spleen cells from rats concomitantly treated with the selective macro-phage toxin silica. in contrast, treatment with d -penicillamine during the last 4 days of silica treatment sometimes resulted in a marked decrease in ³H-TdR incorporation. It is suggested that d -penicillamine treatment in vivo is able to enhance the responsiveness of the lymphocytes, dependent on the presence of functionally intact macrophages. The increased response vanished after 2-3 weeks, even with continuous administration of d -penicillamine.     

Enhanced T Lymphocyte Expression of LFA-1, ICAM-1, and the TNF Receptor Family Member OX40 in HgCl2-Induced Systemic Autoimmunity

Injection of mercuric chloride into Brown Norway (BN) rats induces a T lymphocyte-dependent autoimmune syndrome. In order to investigate whether modification of adhesion and costimulatory molecules on T lymphocytes may be involved in early T lymphocyte activation by HgCl₂, the authors analysed expression of these molecules in peripheral lymph node cells from BN rats at day 4 after injection of HgCl₂. Tri-colour flow cytometry was performed for expression analysis within CD45RC-defined subsets of CD4⁺ and CD8⁺ cells. Compared to control rats, HgCl₂-exposed rats showed increased numbers of lymphocytes, especially of T lymphocyte blast cells. The levels of LFA-1 expression as well as the fractions of ICAM-1⁺ cells were significantly increased in all CD45RC-defined subsets of CD4⁺ and CD8⁺ cells. Within the CD4⁺CD45RC^lo T lymphocyte population, HgCl₂-injected rats showed a highly significant increase in the number of cells expressing OX40, which is a member of the TNF receptor family. Moreover, only CD4⁺CD45RC^lo blast cells of HgCl₂-exposed rats showed decreased expression of CD43, increased expression of CD49d and decreased numbers of CD26⁺ cells. The results indicate that induction of autoimmunity by HgCl₂ in BN rats is associated with altered expression of T lymphocyte costimulatory molecules, predominantly on CD4⁺CD45RC^lo cells, which may be caused by a direct effect of HgCl₂ on these cells, and may precipitate further activation of T and B lymphocytes by HgCl₂     

Effects of Anti-CD 18 and LPS on CD 14 Expression on Human Monocytes

In this study we show that the cytokine stimulatory effect of LPS on human monocytes is enhanced by addition of monoclonal antibodies against CD18 (aCD18 MoAbs). Incubation of monocytes with αCD18 MoAbs overnight increased the CD 14 expression as detected by Leu-M3, but not with My-4. These results indicate that CD18 participates in LPS-induced TNF-o production as well as in regulating CD14 expression on monocytes. Addition of LPS to monocytes resulted in a reduction in the CD14 expression after 1/2, 1, 2 and 4h, but increased CD 14 expression was seen after LPS stimulation overnight. By doing double labelling of the monocyte population for CD 14 and CD16 it was found that the reduction in CD 14 expression occurred in the CD14⁺/CD16⁺ sub-population, while the increase in CD14 expression was seen in both the CD14⁺/CD16⁺ and the CD14⁺/CD16⁺ cells. αCDU MoAbs that were able to inhibit LPS-induced cytokine production from monocytes (3C10 and My-4) were considerably less able to detect the increase in CD14 expression after LPS stimulation than aCD14 MoAbs that did not inhibit LPS-induced cytokine production (Leu-M3 and αCD14_serva)- Our data indicate that My-4 and Leu-M3 define two populations of CD14⁺ cells on LPS stimulated human monocytes.     

Activation of Human Peripheral Blood Lymphocytes: Effect of Concanavalin A and Lipopolysaccharide on in Vitro Synthesis of DNA and Immunoglobulins

We studied the Interaction of lipopolysaccharide (LPS) and concanavalin A (Con A) with regard to IgM and IgG production in in vitro cultures of human peripheral blood lymphocytes (PBL) In our system LPS alone over a wide range Of concentration did not stimulate detectable IgM or IgG production, while Con A at optimal (6 μg ml) and suboptimal (0.6 μg/ml) mitogen concentration induced synthesis of small amounts of Ig. A marked enhancing effect was present when both Con A and LPS were added to the cultures. The different doses of LPS had similar effect on both classes of Ig, and typical dose-response curves were obtained. To evaluate the cellular basis of this synergism, the effect on cell proliferation was studied undo identical experimental conditions in normal subject and patients with X-linked agammaglobulinaemia (X-LA). Parallel cultures were set up after monocyte depletion by adherence on petri dishes. On day 3, increasing doses of LPS were associated with progressive decreases in ³H-thymidine (³H-TdR) Incorporation. Similar result were obtained with normal lymphocytes and those from X-LA patients. Monocyte depletion did not substantially after the lymphocyte response pattern. The preferential Induction of helper activities, either directly by helper stimulation or indirectly by suppressor minimum, is suggested us a possible mechanism of the Interaction observed.     

Mitogenic Activation of Human Lymphocytes: a Protein A Plaque Assay Evaluation of Polyclonal B-Cell Activators

Using the protein A plaque assay, the capacity of various polyclonal B cell activators to induce differentiation in human B lymphocytes was investigated. Dextran sulphate and native Dextran were both virtually devoid of mitogenic properties, Lipopolysaccharide, however, was round to be a potent mitogen in human cells that, although giving rise to low DNA synthetic response, induced high numbers of immunoglobulin-synthesizing cells. Mean plaque-forming cell (PFC) numbers in healthy blood donors assayed on the optimal day (days 5–7) were 23, 493 IgM/10⁴ cells, 11, 288 IgG/10⁶ cells, and 2643 IgA/10⁶ cells. Values obtained in spleen cells, peaking at days 4–6, were slightly higher. Purified protein derivative (PPD) was equally or oven more-effective than lipopolysaccharide (LPS) in generating PFC of different subclasses in peripheral blood with mean of 29, 241 IgM/10⁶, 21, 269 IgG/10⁶, and 3681 IgA/10⁶. PPD furthermore induced a marked DNA synthetic response in human lymphocytes. These data suggest that LPS and PPD may both be used as functional markers in human cells when analysing patients with a suspected immunodeficiency state. It is suggested that cultures should be assayed using the protein A plaque assay, thereby being able not only to investigate the individual immunoglobulin classes but also to avoid the possible hazards involved in measuring antigen-specific responses in patients whose prior immunization to the antigen tested can never be totally excluded     

The Effects of Mitogens, IL-2 and Anti-CD3 Antibody on the T-Cell Receptor Vβ Repertoire

Phytohaemagglutinin (PHA), Concanavalin A (Con A), interleukin-2 (IL-2), and monoclonal antibodies to CD3 (CD3MoAbs) are used for the assessment of the T-cell receptor (TCR) BV gene family expression in autoimmune disorders and multiple sclerosis, and to produce clones for assessment of cytokine profiles in progressive human immunodeficiency virus infection. The authors examined the effects of these stimulants on the TCR Vβ repertoire of resting and blastic CD4⁺ and CD8⁺ normal human peripheral blood lymphocytes, using three-colour cytofluorometry and a panel of anti-TCR Vβ monoclonal antibodies. IL-2 was associated with an increased percentage of blastic CD4⁺ cells expressing V_β5.1 (from median of 3.7% to 8.0%, P  = 0.0002) and blastic CD8⁺ cells expressing V_β5.3 (1.0 to 1.5%, P  = 0.0039). CD3MoAb caused a slight increase in V_β6.7 + blastic CD4⁺ cells (4.5 to 6.9%, P  = 0.0078). PHA did not alter the Vβ repertoire of blastic cells. Con A caused skewing in CD8⁺ blastic cells, toward expression of V_β5.2/5.3 (3.1 to 8.1%) and V_β5.3 (0.8 to 4.8%) ( P  = 0.0020). Thus, IL-2 stimulation causes slight alterations in the Vβ repertoire that should be taken into account in certain research settings. Con A produced skewing in CD8⁺ blastic cells suggesting that, in the presence of CD8, either Con A binds selectively to certain Vβ or the three-dimensional complex created by Con A's binding to other T-cell surface molecules induces preferential V_β5 stimulation.     

Epstein-Barr virus-associated Hodgkin's disease in HTLV-I seropositive patients: A report of two cases

Diagnosis of Hodgkin's disease (HD) is quite difficult in the patient with seropositlvlty for human T cell lymphotropic virus I (HTLV-I). Herein, two cases of Epstein-Barr virus (EBV)-associated HD, which occurred In males with seropositlvity for anti-HTLV-l, are reported. One patient is alive and was diagnosed as having interfollicular HD with CD20⁺CD15^-CD30^-CD3^-CD4^-CD8^-CD45RO^-Reed-Stern-berg (R-S) cells. Positivity for EBV-encoded RNA1 (EBER-1) and latent membrane protein 1 (LMP-1) was shown on folllcular germinal center cells and R-S cells. In that case, neither T cell receptor (TCR) β chain rearrangement nor integration of the HTLV-I provlrus was demonstrated In the lymph nodes, although atyical lymphocytes (2%) were found in the peripheral blood. The other case pursued an aggressive clinical course and the patient was diagnosed as having an adult T cell leukemla/lymphoma (ATLL) because of the presence of antl-HTLV-l antibody, lymph node swelling, and the appearance of flower-like cells in the peripheral blood. However, an autopsy revealed no obvious ATLL cell infiltration in any of the organs examined. Multiple granulomatous lesions were found In the bone marrow, liver, kidneys, spleen, and lymph nodes. Reassessment of lymph node lesions In biopsies and granulomatous lesions in autopsy samples demonstrated that both lesions contained CD15⁺CD30⁺CD3^-CD4^-CD8^-CD20^-CD45RO^-EBER-1⁺LMP-1⁺R-S cells, and they were considered to be a composite lymphoma of HD and ATLL. These two cases therefore suggest that EBV-associated HD can develop in patients with seropositivity for HTLV-I.     

Enumeration of polyclonal mitogen-responsive cells in different lymphoid tissues of the mouse.

The relative number of cells capable of responding to Con A, PHA and LPS in the spleen, blood, lymph node and Peyer's patches of CBA mice has been quantified by means of a cytological analysis technique. No difference has been found between Con A- and PHA-responsive cells in spleen and lymph node. The lymphoid tissues of T cell-deprived mice have a reduced content of PHA responsive cells, but LPS responsiveness is within normal limits. Pretreatment of peripheral lymphocyte populations with high concentrations of anti-O antiserum and complement abolishes the response of the treated cells to PHA, but not to LPS, whereas similar treatment with a cytotoxic anti-immunoglobulin serum, which has no effect on PHA-responsive cells, only partially reduces the response to LPS. The results for mitogen responsiveness are discussed with reference to other methods of quantifying T and B cells using cell-surface markers.     

Alterations in the Mitogen Responsiveness of Lymphocytes from Mice Bearing Moloney Sarcoma Virus Induced Tumors

The effects of Moloney Sarcoma Virus (MSV) induced tumor growth dynamics on the blastogenic responsiveness of lymphocytes from BALB/c mice were investigated. Lymphocytes from spleen, thymus and lymph node pools were tested for responsiveness to phytohemagglutinin (PHA) and concanavalin A (Con A). Results showed a significant decrease in PHA-induced blastogenesis of all lymphocytes tested at the time of maximal tumor volume, with a return to normal responsiveness as the tumor regressed. In contrast, a differential dose dependent Con A response occurred in spleen and thymus lymphocytes. A decreased ³H-thymidine uptake occurred at optimal Con A dose, correlating with the PHA decrease. However, at a lower Con A dose an increased response was observed, beginning shortly before the PHA depression and continuing until regression of tumor began. This phenomena was not observed in lymph node lymphocytes.

Based upon these observations, we suggest that the cell or cells responsible for the transient suppression of PHA responsiveness may be Con A responsive T lymphocytes.     

II. The Effects of Thalidomide Treatment on Autoimmune-Prone NZB and MRL Mice are Consistent with Stimulation of the Central Immune System

We describe here some immunomodulatory effects of thalidomide on autoimmune-prone mice. The highly increased synthesis of splenic IgM in NZB mice, of splenic and lymph node IgG of different subclasses in MRL/n mice, and of splenic and lymph node IgGl in MRL/lpr mice was markedly inhibited by thalidomide treatment. After a single treatment with 3mg of thalidomide, the following changes were observed in NZB mice: (i) an initial decrease in the numbers of large CD5⁺μ^high, and in the numbers of total CD5⁺μ⁻, CD5⁻μ^high, CD5⁺μ^high lymphocyte populations of the pleural cavity followed by a late increase in the numbers of large cells of the three cell populations; (ii) a consistent increase in the numbers of a CD5^lowμ^low pleural lymphoid population; (iii) a consistent reduction in the numbers of splenic large CD5⁺ B cells and an oscillatory increase in the number of cells with CD5⁻ phenotype; (iv) a late reduction in the numbers of splenic total CD5⁺ B cells. These results are consistent with the notion that thalidomide controls a disease-associated expansion of B cells in autoimmune prone mouse strains through a stimulatory effect of the drug on the immune system.     

Progressive Accumulation of Bacterial Lipopolysaccharide In Vivo During Murine Acute Graft-Versus-Host Disease

In a previous report the authors demonstrated that acute graft-versus-host disease (GVHD) was associated with pathologic amounts of tumour necrosis factor alpha (TNF-α) and the appearance of lipopolysaccharide (LPS) in the blood of GVH reactive mice just prior to death. In this study the authors have investigated the kinetics of LPS accumulation in different organs during the course of acute GVHD using a murine model. Unirradiated C57BL/6×AF1 (B6AF₁) mice were transplanted with C57BL/6 (B6) lymphoid cells and killed at predetermined times after transplantation for LPS analysis. Control animals were injected with either 60×10⁶ B6AF1 lymphoid cells (syngeneic) or 60×10⁶ irradiated (2000 rad) CBA lymphoid cells (allogeneic). Lipopolysaccharide began to appear in the liver and the spleen of GVH reactive mice from day 2 post-transplant and by day 10 all GVH reactive mice tested positive for hepatic and splenic LPS. Low levels of LPS were detected in some control mice from days 2 to 10 post-transplant but LPS was never detected after day 10 in control groups. Total hepatic and splenic LPS in acute GVH reactive mice peaked at a time coincident with the appearance of LPS in the serum and with the onset of mortality. These results demonstrate that tissue levels of LPS increase throughout the course of acute GVHD and are sufficient to trigger the release of pathologic amounts of TNF-α from primed macrophages resulting in the cachexia and mortality associated with acute GVHD in this model.     

Gastrointestinal Regulatory Peptides Modulate Mouse Lymphocyte Functions Under Serum-Free Conditions In Vitro

Conditions are described for performing mitogen (Concanavalin A, Con A; lipopolysaccharide, LPS) and mixed lymphocyte reaction (MLR) cultures using serum-free medium. The effects of exogenously adding several gastrointestinal regulatory peptides (β-endorphin, substance P, met-enkephalin, vasoactive intestinal peptide, bombesin and somatostatin) on the incorporation of ³H-methyl-thymidine was determined. It was observed that mitogen stimulation of lymph node cells with Con A was inhibited (70% of control) by vasoactive intestinal peptide (VIP) but spleen cells stimulated by LPS were insensitive to immunomodulation (98% of control). The ability of VIP to inhibit Con A induced thymidine incorporation was concentration dependent (10^-6 to 10^-18 M) and was not attributable to kinetic shifts or cell toxicity. None of the other tested neuropeptides affected Con A or LPS induced blastogenesis. MLR cultures were inhibited by VIP, β-endorphin and somatostatin in a biphasic manner with maximal inhibition observed at 10^-8 to 10^-12 M. Both substance P and bombesin exhibited slight immunoenhancing properties at 10^-14 to 10^-18 M. Met-enkephalin was ineffective as an immunomodulator of MLR cultures. The utility of using serum-free medium in identifying neuropeptides with immunomodulatory properties are discussed.     

Sequential Analysis of Distributions of Donor-derived Thymocytes Bearing T-cell Antigen Receptor (TCR) and Donor-derived la+ Cells in Thymuses of Fully Allogeneic Bone Marrow Chimera in Mice

Lethally irradiated SJL/J mice were reconstituted with B10 bone marrow cells, and the process of thymic reconstitution by donor derived cells positive for I- A or Vβ8 molecules was investigated. The donor-derived la⁺ cells appeared in the medulla on day 7 after reconstitution. The la+ cells became confluent up to day 14, and the cellularity in the medulla on day 17 was almost the same as that in the normal thymus. Dull Vβ8⁺ thymocytes were first recognized in the cortex on day 10 and were identifiable in the medulla by day 14. The Vβ8⁺ cells seemed to be mainly CD4⁺8⁺ double-positive. Furthermore, most of the Vg8'cells in the medulla of chimeras given cyclosporin A for 3 weeks after reconstitution appeared to be CD4⁺8⁺. The present findings demonstrate that CD4 8+ thymocytes which bear a low concentration of TCR exist in the thymic medulla at a relatively early stage when donor-derived la⁺ cells have already settled there. The coincidental appearance and coexistence of la⁺ cells and TCR⁺ thymocytes in the medulla suggest that these histological characteristics may be related to the selection of thymocytes in this area.     

T-ZONE HISTIOCYTES WITH S100 PROTEIN

Histiocytic cells with S100 protein compose a cell lineage independent of the monocyte-macrophage system. Langerhans cells and indeterminate cells in the skin and oral mucosa, interdigitating cells in the T-zone of the lymph node, and other lymphoid tissues belong to this cell lineage. In addition to these cells, small S100⁺ cells showed morphological transition to large histiocytes. In human fetuses, a large number of S100⁺ lysozyme^- NCA⁺ cells first appeared in the thymic medulla by the end of the third month of gestation, and rapidly disseminated to the various lymphoid organs in accordance with the spread of T-lymphocytes during the fourth month of gestation. S100+ small cells were more frequent than large cells and showed more rapid dissemination in the early stage. S100- lysozyme⁺ NGA⁺ immature macrophages appeared in the liver, spleen, lymph node anlage, and other tissues at the second month of gestation, and their distribution was completely different from S100⁺ histiocytes. Fetal development of T-zone histiocytes with S100 protein supported the hypothesis that there are two histiocytic cell lines; one is the monocyte-macrophage system, another is the S100⁺ T-zone histiocyte system.     

Peripheral T-cell Lymphoma of AILD (Angioimmunoblastic Lymphadenopathy with Dysproteinemia) Type Involving Gastrointestinal Tract

A case of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) which showed widespread involvement of the gastrointestinal tract is reported. A lymph node biopsy specimen showed the characteristic histological features of AILD. During the progression of the illness, lymphomatous lesions developed in the gastrointestinal tract, complicated by cytomegalovirus infection. A double immunoenzymatic study using a combination of Ki 67 antibody and antibodies against surface antigens demonstrated that CD3⁺, CD4., and/or T cell receptor (TCR) beta⁺ cells were predominant (67–68%) among the population of proliferating Ki 67⁺ cells, rather than CD8⁺ or CD22⁺ cells. Clonal rearrangement of the TCR beta chain gene was also detected. These findings provide further evidence for the neoplastic nature of lesions of this type, and the diagnosis of peripheral T cell lymphoma.     

Effect of exchange transfusion with Fluosol DA 20% on splenic distribution and immunocompetence of rat lymphocytes

T- and B-splenic lymphocyte frequency, immune response 4 days after immunization with sheep red blood cells (SRBC), and proliferative response to concanavalin A (Con A) were determined 1, 3, and 5 days after exchange transfusion with Fluosol DA 20% (FDA) in adult, male Sprague-Dawley rats vs sham transfused rats. T-cell, T-helper, T-suppressor, and B-lymphocyte were reduced 1 day after transfusion (P less than or equal to 0.001). T- and B-lymphocyte frequencies were still reduced at day 3 (P = 0.0372). By day 5, there were no significant reductions in T-cell, T-helper, T-suppressor, and B-cell frequencies in the FDA-transfused rats. The frequency of cells with cytoplasmic IgG was reduced (P less than or equal to 0.025) in cells harvested from spleens of FDA-transfused rats and tested fresh. Proliferative response of splenic lymphocytes to Con A was unaffected by transfusion with FDA (P greater than or equal to 0.078). Splenic hemolytic plaques in response to SRBC were unaffected if rats were transfused 3 days after immunization with SRBC and 1 day prior to study (P = 0.941), enhanced if rats were transfused 1 day after SRBC immunization and 3 days prior to study (P = 0.0015), and suppressed if rats were transfused 1 day before SRBC immunization and 5 days before study (P less than 0.0001). Transfusion with FDA causes transient decreases in identifiable T and B lymphocytes, depresses cytoplasmic IgG-positive B cells, does not affect proliferative response to Con A, does not affect an ongoing specific immune response, enhances an early specific immune response, and inhibits the induction of a specific immune response.     

The Interferon-α/β Responses of Mice to Herpes Simplex Virus Studied at the Blood and Tissue Level In Vitro and In Vivo

Murine mononuclear leucocytes from bone marrow, spleen, lymph node and blood stimulated in vitro by UV-irradiated Herpes simplex type I virus (HSV) produced about equal proportions of IFN-α and -β determined by immunoassay. Thymocytes produced only IFN-α. The frequency of IFN-α/β mRNA containing cells detected by in situ hybridization was highest with bone marrow (15 per 10⁴ cells), followed by spleen (4/10⁴), lymph node (2/10⁴), blood (1/10⁴) and thymus (0.2/10⁴). Such IFN-α/β producing cells (IPCs) were heavily labelled in autoradiographs, each producing about 0.4 U of IFN. After one intravenous injection of UV-irradiated HSV in mice, high levels of IFN-α and -β were present in blood at 3–9 h and little or none at 24 h or later. Frequent cells strongly positive for IFN-α mRNA at in situ hybridization and for IFN-α/β at immunohistochemical staining were found almost exclusively in the marginal zones of spleens. Occasional IPCs were detected in lymph nodes but not in bone marrow, liver and kidneys. The marginal zone IPCs may be the major source of IFN in blood, and high splenic levels of IFN-α/β should have efficient antiviral and immunoregulatory functions.     

CHANGES IN HISTOLOGY OF THE REGIONAL LYMPH NODES AND IN THE PROPORTIONS OF T AND B CELL POPULATIONS BY OXAZOLONE PAINTING OR LPS INJECTION IN GUINEA PIGS

Changes in the regional lymph nodes from guinea pigs stimulated with oxazolone painting or LPS injection were examined by light and electron microscopy at various intervals. The proportions of T and B cells in the nodes were estimated by rosette formation technique^30,34 and weights of the thymus and spleen were measured at the same time. The first event occurring in the nodes after antigenic stimulation was increase in number and activity of post-capillary venules (PGV) which resulted in augmentation of an influx of small lymphocytes from the recirculating pool. Lymphocytes which streamed into the nodes promptly flowed out through the sinus endothelium into the pool. The second event was specific accumulation of committed lymphocytes³⁵. The proportion of T cell in the node suspension reached a peak 4 days after oxazolone painting when morphological changes became maximum and the thymus enlarged. On the other hand, in LPS group, B cells reached a peak on day 5 or 6 when relatively immature germinal centers increased in the nodes and decreased on day 7 because of their differentiation into plasma cells.     

Macrophage Populations in Different Stages of Induced Hepatic Metastases in Rats: an Immunohistochemical Analysis

Macrophages play a role in the host defence against cancer. Little is known about changes in macrophage populations during early metastatic growth. To evaluate the distribution, number and phenotype of macrophages in the development of hepatic metastases in a rat model (Wag/Rij rats and syngeneic CC531 colon carcinoma cell line), an immunohistochemical study was performed with the monoclonal antibodies ED1 (monocytes, and all macrophages), ED2 (resident tissue macrophages, like Kupffer cells) and ED3 (a subpopulation of macrophages which may play a role in the recruitment of lymphocytes). OX19 and Hisl4 were used to identify lymphocytes. In this study a new monoclonal antibody CC52 is described, which recognizes the CC531 tumour cell line. Liver metastases were induced by injection of CC53I colon carcinoma cells into a mesenteric vein. Rats were killed at various intervals. Results show three major macrophage populations during hepatic tumour growth: (1) on day 3, infiltrates are observed around the micrometastases, which contain mainly newly recruited macrophages (ED1⁺ and ED2⁻); (2) after 7 days, ED3-positive (ED3 ⁺) macrophages together with T lymphocytes are found in the infiltrates; (3) an increase in the number of ED2-positive (ED2⁺) Kupffer cells is observed in the liver parenchyma after 14 days. In conclusion, the present results suggest that various populations of macrophages, newly recruited (ED1⁺) as well as resident Kupffer cells (ED2⁺), are involved in the immune response against tumour cell deposits in the liver.     

Proliferation Kinetics of Rat Kupffer Cells after Partial Hepatectomy Immunohistochemical and UItrastructural Analysis

In an effort to settle the conflicting views on the proliferation kinetics of Kupffer cells (Kc), we performed 2/3 partial hepatectomy on rats injected with Pelikan ink. Using an anti-rat macrophage monoclonal antibody, ED 2, we evaluated the numerical changes in total, carbon-positive ED 2⁺ cells and carbon-negative ED 2⁺ cells in the portal and central area. We also analyzed the ultrastructure and peroxidase cytochemistry of various types of cells observed during regeneration. The total numbers of ED 2⁺ cells in the remaining liver increased rapidly from day 2 to 5, and the number of dividing ED 2⁺ cells reached a maximum on day 2. Thus, the numerical increase in ED 2⁺ cells corresponded to the division phase. In contrast, the carbon-labeling experiment showed a continuous increase of carbon negative ED 2⁺ cells from day 2 to 7. In the central area where division was less frequent, the proportion of carbon-positive cells decreased markedly to 50% on day 7, as against 97% in control rats. These findings suggest the possibility of an influx of carbon-negative Kc in addition to cell division. Ultrastructurally, the presence of carbon-negative "small Kc" and "immature Kc" with morphological features different from those of carbon-positive Kc was demonstrated. Such carbon-negative Kc with a high nucleus-to-cytoplasm ratio and rather few phagosomes, were not observed in control rats. Furthermore, we demonstrated two types of possible precursor cell, i.e. "transitional" forms between monocytes and Kc, and "immature macrophages". The former showed peroxidase activity in some lysosomes as well as in the rough endoplasmic reticulum and nuclear envelope. Our result indicated that the proliferation kinetics of Kc depend upon both local proliferation and influx.