Immunoglobulin G anti-endothelial cell antibodies: inducers of endothelial cell apoptosis in pulmonary arterial hypertension?

Endothelial cell (EC) apoptosis seems to play an important role in the pathophysiology of pulmonary arterial hypertension (PAH). We aimed to test the hypothesis that circulating anti-endothelial cell antibodies (AECA) of PAH patients induce EC apoptosis. Immunoglobulin (Ig)G was purified from sera of PAH patients (n = 26), patients with systemic lupus erythematosus (SLE) nephritis without PAH (n = 16), patients with systemic sclerosis (SSc) without PAH (n = 58) and healthy controls (n = 14). Human umbilical vein endothelial cells (HUVECs) were incubated with patient or healthy control IgG for 24 h. Thereafter, apoptosis was quantified by annexin A5 binding and hypoploid cell enumeration by flow cytometry. Furthermore, real-time cell electronic sensing (RT-CES™) technology was used to monitor the effects of purified IgG from patient and healthy control IgG on HUVECs. As demonstrated previously, IgG of AECA-positive SLE nephritis patients (n = 7) induced a higher percentage of apoptosis of HUVECs compared to IgG of AECA-negative SLE nephritis patients and healthy controls. Furthermore, IgG of AECA-positive SLE nephritis patients induced a marked decrease in cell index as assessed by RT-CES™ technology. IgG of AECA-positive PAH patients (n = 12) and SSc patients (n = 13) did not alter the percentage of HUVEC apoptosis or cell index compared to IgG of AECA-negative PAH and SSc patients and healthy controls. AECA-positive PAH patients, in contrast to SLE nephritis patients, do not have circulating IgG AECA that enhances apoptosis of HUVECs in vitro. Further studies should focus on other mechanisms by which AECA may enhance EC apoptosis in PAH, such as antibody-dependent cell-mediated cytotoxicity.

The influence of afferent lymphatic vessel interruption on vascular addressin expression.

Tissue-selective lymphocyte homing is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. These vessels, the post capillary high endothelial venules (HEV), are found in organized lymphoid tissues, and at sites of chronic inflammation. Lymphocytes bearing specific receptors, called homing receptors, recognize and adhere to their putative ligands on high endothelial cells, the vascular addressins. After adhesion, lymphocytes enter organized lymphoid tissues by migrating through the endothelial cell wall. Cells and/or soluble factors arriving in lymph nodes by way of the afferent lymph supply have been implicated in the maintenance of HEV morphology and efficient lymphocyte homing. In the study reported here, we assessed the influence of afferent lymphatic vessel interruption on lymph node composition, organization of cellular elements; and on expression of vascular addressins. At 1 wk after occlusion of afferent lymphatic vessels, HEV became flat walled and expression of the peripheral lymph node addressin disappeared from the luminal aspect of most vessels, while being retained on the abluminal side. In addition, an HEV-specific differentiation marker, defined by mAb MECA-325, was undetectable at 7-d postocclusion. In vivo homing studies revealed that these modified vessels support minimal lymphocyte traffic from the blood. After occlusion, we observed dramatic changes in lymphocyte populations and at 7-d postsurgery, lymph nodes were populated predominantly by cells lacking the peripheral lymph node homing receptor LECAM-1. In addition, effects on nonlymphoid cells were observed: subcapsular sinus macrophages, defined by mAb MOMA-1, disappeared; and interdigitating dendritic cells, defined by mAb NLDC-145, were dramatically reduced. These data reveal that functioning afferent lymphatics are centrally involved in maintaining normal lymph node homeostasis.

Endothelial progenitor cells are differentially impaired in ANCA-associated vasculitis compared to healthy controls.

BACKGROUND: Endothelial progenitor cells (EPC) are of major importance in vascular repair under healthy circumstances. Vascular injury in need of repair occurs frequently in ANCA-associated vasculitis (AAV). A specialized T cell subset enhancing EPC function and differentiation has recently been described. These angiogenic T cells (Tang) may have an important impact on the vascular repair process. Therefore, the aim of our study was to investigate EPC and Tang in AAV. METHODS: Fifty-three patients suffering from AAV and 29 healthy controls (HC) were enrolled in our study. Forty-four patients were in remission, nine patients were in active state of disease. Patients were either untreated or were under monotherapy with low-dose steroids (max. 5 mg/day) at the time of sampling. Circulating EPC and Tang were determined by flow cytometry (FACS). The functional capacity of EPC was assessed by established cell culture methods. RESULTS: Circulating EPC were significantly decreased in AAV as compared to HC. The capacity of EPC to differentiate and proliferate was differentially impaired in patients as compared to HC. The outgrowth of endothelial colony-forming cells (ECFC) was severely decreased in patients whereas colony-forming units-endothelial cell (CFU-EC) outgrowth was unaffected. ECFC and CFU-EC differentiation was strictly T cell-dependent. Patients with a relapsing disease course had an impaired ECFC outgrowth and expansion of Tang as compared to patients with a stable, nonrelapsing disease. CONCLUSIONS: The differentiation process of EPC is impaired in AAV. This may favor insufficient vascular repair promoting a relapsing disease course. Finally, these factors may explain a higher cardiovascular morbidity as has been previously documented in AAV.

Identification of the bone marrow-derived ia positive cells in the rat thymus: a morphological and cytochemical study.

Ia+ cells of bone marrow origin were studied in the thymus of normal and bone marrow-reconstituted radiation chimeras of rats and characterized on morphology and acid phosphatase (APh)- and Ia-staining pattern in-vivo and in-vitro. In-vivo, the majority of the bone marrow-derived Ia+ cells were present in the medulla. They were large, had cell processes extending between surrounding thymocytes, and frequently weak APh activity. In-vitro, the Ia staining was demonstrated on the cell surface in variable intensity. Ia+ cells showed a frayed outline with cell processes between adherent thymocytes and had weak cytoplasmic APh activity, frequently in a central spot. They had an elongated, usually invaginated, nucleus and a rather pale cytoplasm, in which Birbeck granules sometimes were observed. The characteristics of the bone marrow-derived Ia+ cells demonstrate these cells are an equivalent of the interdigitating cells present in thymus medulla. They distinguish themselves from a population of Ia-, strongly APh+ macrophages predominantly localized in the cortex, and from the Ia+, APh- thymic reticular epithelial cells.

Lymphocyte migration into the skin: the role of lymphocyte homing receptor (CD44) and endothelial cell antigen (HECA-452).

Lymphocyte migration into the lymphoid organs and sites of inflammation is controlled by lymphocyte-endothelial cell interaction at sites where lymphocytes exit from the blood. Expression of Hermes-defined CD44 class of lymphocyte homing receptor and HECA-452 antigen specific for high-endothelium-mediating physiologic lymphocyte extravasation was studied in dermatitis herpetiformis, celiac disease, psoriasis, mycosis fungoides, lymphocytosis cutis, atopic dermatitis, and allergic contact dermatitis. Also, duodenal biopsies of patients suffering from dermatitis herpetiformis or celiac disease were studied for existence of these antigens. Infiltrating lymphocytes in the skin and in the duodenal area expressed homing receptor molecules when studied with monoclonal antibodies, Hermes-1 and Hermes-3, that recognize the CD44 class of molecules involved in lymphocyte binding to high endothelial venules in peripheral lymph nodes, mucosa-associated lymphatic tissues, and inflamed synovium. However, the HECA-452 antigen was not detected on the venules, neither in the skin nor in the duodenum. Even the venules possessing high endothelium morphologically were HECA-452 negative. These findings suggest the CD44 class of lymphocyte homing receptor(s) is also involved in lymphocyte homing to inflamed skin and the duodenal area of the gut. However, on the basis of HECA-452 staining, high endothelial venules in inflamed skin and duodenum are not antigenically identical with high endothelial venules in organized lymphoid tissues. This finding indirectly supports the idea that molecules and/or mechanisms mediating lymphocyte extravasation might be distinct in these organs.

Renal injury and salt-sensitive hypertension after exposure to catecholamines.

We investigated whether chronic infusion of phenylephrine could induce structural and functional changes in the kidney of rats with the subsequent development of salt-sensitive hypertension. Rats were infused with phenylephrine (0.15 mmol/kg per day) by minipump, resulting in a moderate increase in systolic blood pressure (BP) (17 to 25 mm Hg) and a marked increase in BP variability as measured by an internal telemetry device. After 8 weeks, the phenylephrine infusion was stopped with the return of BP to normal, and a nephrectomy was performed for histological studies. Glomeruli were largely spared, but focal tubulointerstitial fibrosis was present, with the de novo expression of osteopontin by injured tubules, macrophage and "myofibroblast" accumulation, and focal increases in mRNA for transforming growth factor beta by in situ hybridization. Peritubular capillaries at sites of injury had distorted morphology with shrinkage, rounding, and focal rarefaction, and endothelial cell proliferation was also identified. Rats were randomized to a high (8% NaCl or 1.36 mol/kg) or low (0.1% NaCl or 17 mmol/kg) salt diet. After 4 to 8 weeks, phenylephrine-treated rats on a high salt diet developed marked hypertension, which was in contrast with phenylephrine-treated rats placed on a low salt diet or vehicle-treated rats given a high salt diet. Hypertension after phenylephrine exposure correlated with the initial mean systolic BP (r(2)=0.99) and the degree of BP lability (r(2)=0.99) during the phenylephrine infusion, the amount of osteopontin expressed in the initial biopsy/nephrectomy (r(2)=0.74), and the final glomerular filtration rate (r(2)=0.58). These studies provide a mechanism by which a markedly elevated sympathetic nervous system can induce salt-dependent hypertension even when the hyperactive sympathetic state is no longer engaged.

Comparison of detection techniques for cytokine reverse transcriptase polymerase chain reaction; digoxigenin-labeled polymerase chain reaction permits sensitive detection of cytokine mRNA in rat heart allografts.

The polymerase chain reaction (PCR) is a sensitive method for the analysis of cytokine mRNA expression. The amount of specific mRNA in tissues involved in an inflammatory immune response can be low and therefore requires highly sensitive detection of the PCR products. In our study we have compared different detection techniques in order to replace the commonly used detection by means of radiolabeled probes. Besides the detection of DNA in agarose gels by ethidium bromide (EB), we used detection by digoxigenin (DIG)-labeled probes, as well as the direct incorporation of DIG-labeled nucleotides in the PCR, in comparison to detection by means of 32P-labeled probes. In vitro activated rat lymph node cells, lymph node tissue, and acutely or chronically rejected rat heart allografts were examined for expression of mRNA of the cytokines IL-2 and IFNgamma. The directly DIG-labeled PCR appeared to be the best alternative for detection of PCR products by means of radiolabeled probes. While IL-2 mRNA was not detected by means of EB and IFNgamma mRNA was only detected at the highest PCR cycle numbers in acutely and chronically rejected rat heart allografts, both cytokine mRNA's were readily detected by directly DIG-labeled PCR.

Chronic renal allograft rejection. Selective involvement of the glomerular endothelium in humoral immune reactivity and intravascular coagulation.

To study immune reactive and thrombotic mechanisms involved in chronic renal allograft rejection, Lewis rat kidneys were transplanted into bilaterally nephrectomized Brown Norway recipients tolerant of LEW erythrocyte antigens. Such BN rats fail to produce anti class I MHC alloantibodies after insertion of a LEW kidney. The LEW renal allografts experience a transient rejection episode without proteinuria followed by the development of chronic rejection, clinically characterized by glomerular proteinuria in the presence of stable renal function. Immunohistological studies of such chronically rejected LEW renal allografts showed the occurrence of glomerular and interstitial infiltration of predominantly monocytes and T cells. CD4-positive T cells dominated over CD8-positive T cells in the chronically rejected LEW renal grafts. IgG deposition was found deposited throughout the renal vasculature--this in contrast to IgM, which was observed only in the glomerular vasculature. Glomerular antibodies were not directed to endothelial class II MHC antigens, and showed only weak complement fixation as demonstrated by C3 staining. Selective glomerular IgM deposition was associated with vascular (platelet-containing) thrombi, and focal and segmental fibrinoid necrosis. In contrast, acutely rejected LEW renal grafts in unmodified BN recipients showed IgM deposition as well as thrombus formation throughout the entire renal vasculature. The results demonstrate that the antibody response to endothelial--and, in particular, glomerular endothelial non-MHC antigens--may bring about chronic vascular renal allograft rejection. How the formation of glomerular thrombotic lesions may be assisted by endothelial reactivity to cytokines from local immune reactive cells is discussed.

Effects of antibody reactivity to major histocompatibility complex (MHC) and non-MHC alloantigens on graft endothelial cells in heart allograft rejection.

BACKGROUND: To gain insight in the pathogenesis of vascular lesions in heart allograft rejection, we investigated effects of allosera reactive with major histocompatibility complex (MHC) or non-MHC alloantigens on graft endothelial cells (EC) in a rat transplantation model. METHODS: Anti-MHC and anti-non-MHC allosera were obtained from Brown Norway (RT.1(n)) recipients of a Lewis (RT.1(1)) or congenic LEW.1N (RT.1(n)) heart allograft respectively. Reactivity with endothelial alloantigens was studied in vitro using a series of three rat heart endothelial cell (RHEC) lines of Lewis origin. Phenotypic studies of MHC and non-MHC alloantigen expression, and adhesion molecule induction on EC were performed by immunostaining and fluorescence-activated cell sorting analysis. Complement-mediated cytotoxicity of allosera was studied using a 51Cr release assay. RESULTS: Both anti-MHC allosera and anti-non-MHC allosera showed reactivity with all three RHEC lines. EC stimulation with tumor necrosis factor-alpha and interferon-y resulted in increased reactivity of anti-MHC but not of anti-non-MHC allosera. Anti-MHC allosera showed complement-mediated cytotoxicity for EC, which was strongly increased when cytokine-stimulated EC were used. With anti-non-MHC allosera, only minor cytotoxicity was measured, irrespective of the activation of EC. Anti-MHC and anti-non-MHC allosera from the day of rejection (days 7-8 and days 29-35, respectively) had similar subclass profiles of allospecific IgG, except for allospecific IgM, which was only detected in anti-MHC allosera. Complement-mediated cytotoxicity of anti-MHC allosera from the day of rejection was effected mainly by IgM alloantibodies, whereas, in allosera taken 4 days after rejection, a predominance of cytotoxic alloantibodies of the IgG class was observed. No indications were found that either alloantibody reactivity alone or in combination with complement activation led to EC activation processes relevant to intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 induction. CONCLUSIONS: Our data show that, in heart allograft rejection, MHC but also non-MHC alloantigens on EC are target structures in the alloantibody response. Alloantibodies reactive with endothelial MHC, but not those reactive with non-MHC alloantigens, may significantly contribute to vasculopathy by complement-mediated cytotoxicity. Although no evidence was found that alloantibodies reactive with graft EC induce adhesion molecule expression, they may trigger other EC mechanisms relevant to graft vasculopathy.

A dual role for endothelial cells in cytomegalovirus infection? A study of cytomegalovirus infection in a series of rat endothelial cell lines.

Several clinical findings point to the involvement of microvascular endothelial cells in cytomegalovirus-related pathology. In this study the interactions of cytomegalovirus (CMV) with microvascular endothelial cells was investigated in an in vitro rat model. A series of rat endothelial cell lines, considered representative for the heterogeneity of heart microvascular endothelium in vivo, were infected with rat CMV (RCMV). The course of infection and production of infectious virus were examined using immunofluorescence staining and plaque titration assays, and was compared with infection of fully permissive rat fibroblasts. These endothelial cell lines displayed differences in susceptibility to CMV infection. Two endothelial cell lines (RHEC 50 and 191) were practically non-permissive, while four endothelial cell lines (RHEC 3, 10, 11 and 116) were partly permissive for CMV infection. In contrast to CMV infection in fibroblasts, only limited infection of the permissive endothelial cell lines was observed without spreading of CMV infection through the monolayer, although infectious virus was produced. Detachment of infected endothelial cells and recovery of the monolayer with time was observed. The detached endothelial cells were able to transmit CMV infection to fibroblast monolayers, but not to endothelial monolayers. Our in vitro results demonstrate differences in permissiveness for RCMV between the series of rat endothelial cell lines, which is suggestive for endothelial heterogeneity to CMV infection in vivo. Our findings indicate that endothelial cells are relatively resistant to CMV infection and that, upon infection, the endothelial monolayer may dispose of the virus via detachment of the infected cells. This points to a dual role for the endothelium in CMV infection in vivo: a barrier for CMV infection (by the endothelial monolayer) on the one hand and spreading of CMV infection (by detached infected cells) on the other hand.

The superantigen exfoliative toxin induces cutaneous lymphocyte-associated antigen expression in peripheral human T lymphocytes.

Several immune-mediated dermatoses including psoriasis and atopic dermatitis can be exacerbated by bacterial infections. Superantigen producing bacteria can be isolated from skin lesions of these dermatoses. Consistent with superantigen effects, skewed T cell receptor variable gene usage has been demonstrated within these lesions. Therefore, the question arises whether superantigen induce a skin-seeking phenotype within peripheral T cells. In this study, we investigated the in vitro influence of the V beta 2-selective superantigen exfoliative toxin from Staphylococcus aureus on the expression of the cutaneous lymphocyte-associated antigen on peripheral T lymphocytes of healthy donors. We demonstrate that exfoliative toxin dramatically upregulates cutaneous lymphocyte-associated antigen expression on T cell receptor V beta 2+ lymphocytes. Up to 69% of V beta 2+ lymphocytes expressed cutaneous lymphocyte-associated antigen after 5 days of in vitro culture. Additionally, exfoliative toxin also increased cutaneous lymphocyte-associated antigen expression in CD3+ T cell receptor V beta 2- lymphocytes indicating a different effect as caused by the superantigen-T cell receptor V beta 2 interaction. Our findings suggest influence of bacterial superantigens on T lymphocyte skin homing in vivo.

Ontogeny of the rat thymus micro-environment: development of the interdigitating cell and macrophage populations.

The ontogeny of the rat thymus micro-environment and in particular the development of the interdigitating cell (IDC) and macrophage (M phi) populations has been studied. At day 15 of fetal life the thymus consisted of an epithelial primordium in which some Thy-1 positive thymocytes were present around local capillaries in a central area. At day 16 some Ia positive cells, which could not be further identified, and some monocyte-like M phi were observed in the central area. From day 17 the thymus became lobulated by ingrowth of small blood vessels with perivascular connective tissue from the surrounding capsule. An Ia positive epithelial reticulum developed which became populated by increasing numbers of thymocytes. Some strongly acid phosphatase positive M phi were present from this stage of development. From day 19 cortical and medullary areas could be distinguished in the thymus. The cortex consisted of an Ia positive epithelial reticulum in which closely packed thymocytes and scattered M phi were present. The medulla demonstrated a confluent Ia staining and consisted of an epithelial reticulum in which thymocytes, strongly non-specific esterase positive IDC and an occasional M phi were present. Also highly phagocytic IDC-like cells were observed in the medulla, most likely they comprise the population of differentiating IDC. Thymocyte proliferation areas, which were strongly pyroninophilic, were observed from day 21 in the cortex, just beneath the surrounding connective tissue capsule. A distinct cortico-medullary region with many M phi was present one week after birth. From this stage the IDC and M phi distribution was comparable with older thymi.

Ontogeny of the rat thymus micro-environment: development of the interdigitating cell and macrophage populations.

The ontogeny of the rat thymus micro-environment and in particular the development of the interdigitating cell (IDC) and macrophage (M phi) populations has been studied. At day 15 of fetal life the thymus consisted of an epithelial primordium in which some Thy-1 positive thymocytes were present around local capillaries in a central area. At day 16 some Ia positive cells, which could not be further identified, and some monocyte-like M phi were observed in the central area. From day 17 the thymus became lobulated by ingrowth of small blood vessels with perivascular connective tissue from the surrounding capsule. An Ia positive epithelial reticulum developed which became populated by increasing numbers of thymocytes. Some strongly acid phosphatase positive M phi were present from this stage of development. From day 19 cortical and medullary areas could be distinguished in the thymus. The cortex consisted of an Ia positive epithelial reticulum in which closely packed thymocytes and scattered M phi were present. The medulla demonstrated a confluent Ia staining and consisted of an epithelial reticulum in which thymocytes, strongly non-specific esterase positive IDC and an occasional M phi were present. Also highly phagocytic IDC-like cells were observed in the medulla, most likely they comprise the population of differentiating IDC. Thymocyte proliferation areas, which were strongly pyroninophilic, were observed from day 21 in the cortex, just beneath the surrounding connective tissue capsule. A distinct cortico-medullary region with many M phi was present one week after birth. From this stage the IDC and M phi distribution was comparable with older thymi.

Relation between langerhans cells, veiled cells, and interdigitating cells.

Tissue macrophages are bone marrow derived mononuclear cells which play an important role in the immune response, especially as antigen presenting cells. They comprise a heterogeneous population of cells with phagocytic activity. On morphological functional and cytochemical criteria it is likely that the Langerhans cell (LC) in the epidermis, the veiled cell (VC) in the afferent lymph and the interdigitating cell (IDC) in the thymus dependent area of peripheral lymphoid organs and the thymus medulla belong to a subpopulation of the macrophages. They are low phagocytic, Ia positive and are highly immunogenic. VC and IDC may contain Birbeck granules, the characteristic organelles of the LC, suggesting a relationship between these cell types. An epithelial micro-environment as present in the skin epidermis and the thymus is necessary for the induction of these granules, which appear to have no immunological significance. In a scheme the development from monocyte into LC or into VC and subsequently IDC is postulated. Probably VC transport antigen from the skin area via the afferent lymphatics into the draining lymph node. In the thymus dependent area of this organ they present this antigen to T cells and mature into IDC. IDC in the medullary area of the thymus may also be involved in antigen presentation to immunocompetent T cells. However, in this central lymphoid organ a function in instruction of helper T cells may not be excluded.

Functional capacities of high endothelial venules appear not to be controlled by recirculating lymphocytes.

The influence of recirculating lymphocytes on the function and morphology of high endothelial venules (HEV) has been studied. Mice were depleted of lymphocytes by lethal (1200 cGy) total body irradiation; subsequently, the HEV in mesenteric and cervical lymph nodes were studied up to 7 days after irradiation for: 1) capacity to bind lymphocytes by using the in vitro HEV-binding assay, 2) for morphological aspects such as ultrastructure and endothelial height, 3) for presence of RNA (pyroninophylia) and MECA-325 expression. Although, commencing 3 days after irradiation, lymphocyte depletion was intense and no extravasation of lymphocytes was observed; HEV were capable of binding lymphocytes at normal levels. Also the ratio of B/T cell binding to HEV was comparable to normal. MECA-325 expression, pyroninophilia, and ultrastructure of high endothelial cells were not affected by lymphocyte depletion. However, the average height of endothelial cells, which is a measurement related to cell volume, declined during lymphocyte depletion, stabilizing at about 70% of normal levels from day 4. After intravenous injection of viable lymph node cells, endothelial cell height rapidly increased within a few hours in conjunction with lymphocyte extravasation and homing into the nodes. Restoration of endothelial cell height was not observed after infusion of thymocytes, lethally irradiated lymph node cells or supernatants rich in cytokines. We conclude that recirculating lymphocytes in blood and lymphoid tissues are not involved in controlling high endothelial cell activity including the specific function in lymphocyte extravasation. However, recirculating/extravasating lymphocytes contribute to the development of endothelial cell height. The significance of non-lymphoid (radioresistant) cells in the control of characteristic high endothelial function is suggested.

Heart EC respond heterogeneous on cytokine stimulation in ICAM-1 and VCAM-1, but not in MHC expression. A study with 3 rat heart endothelial cell (RHEC) lines.

Cytokine-induced expression of ICAM-1, VCAM-1, and MHC class I and II was studied at different time points in microvascular endothelial cells (EC) of heart origin, using three different rat endothelial cell (RHEC) lines that were stimulated with TNFalpha and/or IFNgamma. Each of the three RHEC lines responded to TNFalpha as well as to IFNgamma; stimulation with combined cytokines led to increased or even synergistic effects. TNFalpha was most potent in inducing ICAM-1 and VCAM-1, whereas MHC class II was most effectively induced by IFNgamma. The 3 RHEC lines responded similarly regarding induction of MHC class II and upregulation of constitutively expressed MHC class I on the cells. However, the RHEC lines showed remarkable differences with respect to ICAM-1 and VCAM-1 induction, with each line having a unique expression profile. In RHEC-3, both ICAM-1 and VCAM-1 were well inducible, whereas in RHEC-10, no ICAM-1 and only some VCAM-1 could be induced. RHEC-11 showed minimal induction of ICAM-1, but strong induction of VCAM-1. For P-selectin induction, no such differences were found between the RHEC lines. These heterogeneous effects of cytokine stimulation could neither be explained by differences in mobilization of calcium nor by ultra-structural differences between the lines. Stimulation of the RHEC lines for ICAM-1 and VCAM-1 or MHC class II molecule induction resulted in expressing and non-expressing EC. Experiments with selected and subsequently cultured expressing and non-expressing cell populations for either ICAM-1, VCAM-1 or MHC class II, indicated that this selective induction most likely results from intrinsic regulation mechanisms in the cell cultures, and not from the presence of particular EC subpopulations within the lines. We conclude that microvascular heart endothelial cells, as represented by the 3 RHEC lines, demonstrate a selective heterogeneity in expression of ICAM-1 and VCAM-1, but not of MHC class I and II, upon cytokine stimulation. The consequences of this heterogeneity for leukocyte-endothelial cell interactions in heart inflammation and immune reactivity is discussed.

Reactivation of rat cytomegalovirus in lung allografts: an experimental and immunohistochemical study in rats.

Reactivation of latent rat cytomegalovirus (RCMV) from a lung allograft or from a recipient was studied in RCMV-mismatched combinations (donor [D]-/recipient [R]+, D+/R-, and D+/R+) with latently infected lung grafts and chronically infected rats in an inbred rat model. Nineteen transplants in a major histocompatibility complex different strain combination (Brown-Norway/Lewis) were immunosuppressed daily (cyclosporine, azathioprine, and prednisolone) from day 3 after orthotopic left lung transplantation and killed on days 3, 6, and 21. Control groups consisted of nine chronically RCMV-infected rats with immunosuppression without transplantation and six allografts with immunosuppression without RCMV infection. Reactivation of latent RCMV was tested by immunohistochemical staining with monoclonal antibodies against RCMV-induced antigens and by plaque assays of the virus in the salivary glands. The following results were obtained: (1) All allotransplants developed acute ongoing rejection on days 3 and 6, and the rejection was resolved on day 21 by immunosuppression. (2) Reactivation was observed in allotransplanted groups, but not in the control rats. (3) In the D+/R+ and D-/R+ groups on days 3 and 6, the number of RCMV-related antigen-positive cells increased in the recipient spleen and lymph nodes and in the bronchus-associated lymphoid tissue of the donor lung in the D-/R+ group, but not in the chronically RCMV-infected controls. (4) In the D+/R- group on day 6, RCMV-induced antigen-positive cells were observed in the spleen and lymph nodes of the recipient and also around the vessels in the recipient lung. (5) In the D-/R+ group, vascular endothelial cells or mildly infiltrated mononuclear cell subpopulations around the vessels in the lung allograft showed weakly positive staining against RCMV-related antigens on day 6. (6) After the initial acute rejection on days 3 and 6 was treated by immunosuppressive drugs, reactivated acute RCMV infection became chronic or latent again on day 21. We conclude that RCMV infection could be transferred with latently infected lung allografts by reactivation of latent RCMV. In rats, as in man, alloimmune responses seemed to have a definite influence on the reactivation of latent RCMV after lung transplantation.

Immunohistochemistry of Nasopharyngeal (Waldeyer's ring equivalent) lymphoid tissue in the rat.

Previously we have described the presence of Waldeyer's ring equivalent (WRE) lymphoid tissue in the rat, and pointed out the importance of such an experimental model for studying the immunological role of nasopharyngeal lymphoid tissue. Here we extend this work with immunohistological data in terms of compartmentalization and distribution of the various lymphoid and non-lymphoid cells of this WRE lymphoid tissue in situ. WRE tissue consists of distinct T cell and B cell areas. B cell areas predominate; they are located directly under the mucosal epithelium and consist mainly of follicles. These follicles frequently contain a germinal center with IgD negative B cells interspersed with scattered CD4 (helper/inducer) T cells. Follicular dendritic cells are present in the germinal centers. T cell areas, on the other hand, are predominantly present at the abluminal side of the WRE in interfollicular areas. In these areas high endothelial venules and both CD4 and CD8 (suppressor/cytotoxic) T cell populations with a clear preponderance of CD4 over CD8 cells can be observed. MHC class II positive interdigitating dendritic cells are also scattered throughout these T cell areas. Mononuclear phagocytes (ED1-positive monocytes/macrophages) are scattered throughout the WRE, but especially in the T cell areas. A subpopulation of (ED3-positive) mononuclear phagocytes, e.g., the lymphoid tissue macrophage, is exclusively scattered between the small blood vessels along the abluminal side of the lymphoid tissue. Here, plasma cells, including those of the IgA type, are located. The data show that nasopharyngeal lymphoid tissue in the rat can be considered as an immunologically fully equipped and active mucosal lymphoid organ presumably executing similar immune functions as the tonsils in the human Waldeyer's ring.