The protein kinase C system in focal adenitis of the lacrimal gland in the non-obese diabetic mouse model for Sjögren's syndrome.

PURPOSE: Non-obese diabetic (NOD) mice develop an autoimmune exocrinopathy characterized by hyposecretion of saliva and acinar cell atrophy. As the protein kinase C (PKC) system is involved in the signal transduction pathways associated with primary secretion and acinar cell differentiation and growth, the PKC profile was analysed in NOD mice. METHODS: Lacrimal glands from BALB/c, NOD, NOD scid and transgenic NOD x interferon-gamma (IFN-gamma) mice were analysed for their PKC profiles using antibodies against several conventional (alpha, beta, gamma), novel (delta, epsilon, theta) and atypical (iota, lambda) PKC isoforms using the Streptavidin/HRP (horseradish peroxidase) method. RESULTS: Acinar cells in BALB/c control mice expressed two conventional (alpha, beta) and two atypical (iota, lambda) PKC isoforms. In NOD and transgenic NOD x IFN-gamma mice the same isoforms were more strongly expressed. NOD scid mice lacked all other PKC isoforms except PKC lambda. CONCLUSIONS: Co-expression of several PKC isoforms in single cell type may be necessary for transcriptional activation and agonist-induced secretory responses. Hyposecretion in NOD mice was paradoxically associated with up-regulation of the PKC system. This may be associated with a deranged signal transduction per se rather than with the immune-inflammation, as the transgenic NOD x IFN-gamma mice showed similar PKC profiles. The NOD model does not reproduce lack/consumption of PKC II and PKC as in Sjögren's syndrome. This may be because the receptor autoantibodies in mice are directed against the adrenergic, not muscarinic, receptors. Lack and/or low level PKC expression in NOD scid mouse may explain the excessive acinar cell apoptosis in this model.

Immunohistochemical study of necrotizing lymphadenitis: a possible mechanism for apoptosis involving perforin and granzyme-producing cytotoxic T cells.

Twelve lymph node specimens with necrotizing lymphadenitis and which had florid necrotic lesions were studied immunohistochemically. The majority of viable lymphoid cells in the necrotic foci were CD8+ lymphocytes and KP1+ or PGM1+ phagocytizing macrophages. The CD8+ T cells were Leu1+, Leu2+, Leu3-, Leu4+, Leu5b+, Leu7-, Leu11b- and Leu19-, indicating a suppressor/cytotoxic T cell phenotype. In addition, the cytoplasm of these cells was immunoreactive for perforin and granzyme B in a granular pattern. With a nick end-labeling technique, fragmented nuclei and some lymphoid cell nuclei were positively stained. These results suggest that the necrosis in necrotizing lymphadenitis is apoptotic necrosis of T cells targeted by CD8+, perforin and granzyme-producing, activated cytotoxic T cells, supporting a viral infection etiology.

Increased activities of cytosol aminopeptidase and lactate dehydrogenase in serum originate from lymphocytes in necrotizing lymphadenitis.

In three pediatric patients with necrotizing lymphadenitis, cytosol aminopeptidase activity (c-AP; EC 3.4.11.1) in serum was markedly increased to 509, 417, and 191 U/L, respectively (normal range 25-60 U/L). Lactate dehydrogenase (LD; EC 1.1.1.27) was also increased, with LD-3 predominating. The increased concentrations of c-AP and LD presumably originated from the destruction of infected, activated lymphocytes, especially T lymphocytes. Necrotizing lymphadenitis is probably caused by a lymphocytotropic virus.

Expression of enolases in B cell tumors.

Frozen lymph node biopsy specimens from 38 patients with B cell tumors, including 5 with childhood non-T-ALL and 3 with reactive lymphadenitis, were investigated using a direct immunohistochemical method to detect alpha-, beta- and gamma-enolases. alpha-Enolase-positive cells were not observed in reactive lymphadenitis. On the contrary, almost all the lymphocytes including germinal center cells were positive for beta-enolase. Small lymphocytes in the mantle zones were negative, centrocytes were negative or weakly positive, the majority of centroblasts were strongly positive and the remaining were weakly positive for gamma-enolase. In all 5 patients with childhood non-T-ALL, leukemic lymphocytes were strongly positive only for alpha-enolase. In all 33 patients with B cell lymphoma, lymphoma cells were positive for beta-enolase. In many patients with follicular lymphoma, lymphoma cells were positive only for beta-enolase. Four of five patients with malignant lymphoma, diffuse, small cleaved cell, showed the reactivity of alpha-, beta+, gamma+-enolases in lymphoma cells. Our results suggest the possibility of the two isoenzyme switches from alpha- to beta-enolase and from alpha- to gamma-enolase in the B lymphocyte lineage accompanying differentiation, similar to those of skeletal muscles and neurons.

Enzyme- and immunohistochemical study of a case of histiocytic necrotizing lymphadenitis.

A combined morphological, immunohistological, and enzyme histochemical analysis was performed on frozen and fixed lymph node tissue in a case of histiocytic necrotizing lymphadenitis (HNL) using conventional histology, a panel of monoclonal and polyclonal antibodies, and a series of common haematological enzyme reactions. Histology showed multiple paracortical necrotizing foci which, in a prominently necrobiotic background devoid of granulocytes, contained large numbers of foamy histiocytes and macrophages intermingled with cells resembling degenerating plasmacytoid T-cells. Most of the histiocytes were alpha1-antichymotrypsin positive and foamy cells were also distinctly Leu-M1 positive. Strong granular acid phosphatase (AP) positivity was present in the cytoplasm of the macrophages and histiocytes. The cells with plasmacytoid features showed weaker and homogeneously diffuse AP staining. Alpha-naphthyl acetate esterase (ANAE) activity was much less striking than AP in the necrotizing foci and most of the ANAE negative cells corresponded to those with plasmacytoid features. No cells with B-cell lineage markers were present within the necrotizing foci; most of the occasional T-cells (Leu-1+, Leu-4+) present in the foci were Leu-2a+ (OKT8+) whereas OKT10+ lymphoid cells were abundant and appeared to correspond with the cells with plasmacytoid features. Our combined data confirm that the special type of necrosis found in HNL develops within foci of plasmacytoid T-cells undergoing regressive changes and apparently exhibiting distinct immunohistological and enzyme histochemical features.

Immunohistochemical localization of alpha 1-antitrypsin and alpha 1-antichymotrypsin in salivary pleomorphic adenomas.

Immunohistochemical identification of alpha 1-antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACh) in pleomorphic adenomas of salivary glands is reported in order to compare their distribution profiles with those of lysozyme and lactoferrin, already described elsewhere. Normal salivary glands indicated positive alpha 1-AT staining in ductal segments and had no alpha 1-ACh in any glandular cell. Pleomorphic adenomas displayed moderate positivity to alpha 1-AT staining in duct-like, tubular and glandular epithelia which was particularly intense in luminal cells. The limited number of tumour cells which showed duct-like structures with a single cellular layer arrangement, displayed the highest staining to alpha 1-ACh. Strongly alpha 1-AT positive tumour cells located on the inner side of luminal cavities were also markedly positive to alpha 1-ACh. Spindle shaped tumour cells existed outside tubular and ductal structures and were negative to alpha 1-AT and alpha 1-ACh. Distribution of alpha 1-AT in salivary glands was similar to that of lysozyme as is usual in ductal segments or their transformed cells, and occurrence of alpha 1-ACh localization rather resembled that of lactoferrin, with occurrence in acinar compartments and changed epithelia within acini. The biological role of a specific immunohistochemical distribution of alpha 1-AT and alpha 1-ACh in pleomorphic adenomas may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases.

The paracortical area in reactive lymph nodes demonstrating sinushistiocytosis. An enzyme- and immunohistochemical study.

The enzyme and immunohistochemical features of lymphnodes showing sinus histiocytosis have been studied. Sinus histiocytes with phenotype OKM1+ OKT4+ Leu3a+ To5+ OKIal- showed strong acid phosphatase and non-specific esterase, weak endogenous peroxidase and no ATPase activities. In nine out of ten lymph nodes, paracortical collections of dendritic OKT6+ OKIal+ cells were observed. In two of the four cases studied these dendritic cells showed strong ATPase activity. We suggest that the dendritic OKT6+ OKIal+ ATPase+ interfollicular cells represent newly arrived veiled cells (VC) which have entered the lymph node by the afferent lymph, settled in the interfollicular area and are probably involved in the induction of a cellular immune response. OKT6+ OKIal+ ATPase+ VC may subsequently transform into mature, OKT6- OKIal+ ATPase+ interdigitating reticulum cells which are involved in the negative feedback of the cellular immune response. The association with sinus histiocytosis is probably related to the fact that an increase in mononuclear phagocytes in the afferent lymph is accompanied by a relative increase in VC. Our results demonstrate that in lymph nodes showing sinus histiocytosis, two cell types increase in number, i.e. an Ia- sinusoidal cell, engaged in phagocytosis of foreign material, and an Ia+ dendritic cell in the interfollicular area, probably involved in the induction of a cellular immune response.

Immunohistochemical demonstration of lysozyme in normal, reactive and neoplastic cells of the mononuclear phagocyte system.

Using the peroxidase antiperoxidase (PAP) method, lysozyme (LZM) was shown to exist in normal, reactive and neoplastic cells belonging to the mononuclear phagocyte system (MPS), but was not detected in histiocytosis X cells. Immunostaining for cytoplasmic LZM by the PAP method is useful for identification of mononuclear phagocytes and for diagnosis of the diseases in which these cells participate.

Alkaline phosphatase positive lymphoma.

Alkaline phosphatase (AP) activity on cryostat sections of the skin of 38 cases of non-Hodgkin's lymphomas of the B cell type and of 10 cases of lymphadenosis benigna cutis have been studied. Membrane-bound AP activity has been found in 8 out of 28 cases of low-grade malignant lymphomas of the B cell type and in 2 out of 10 cases of lymphadenosis benigna cutis. This is a known phenomenon in the latter. In the literature of non-Hodgkin's lymphoma AP activity is related to the intermediate type of lymphocytic lymphoma. Further studies are needed to demonstrate that the AP activity correlates with a specific cell type in the B lymphoid cell differentiation.

[Serum activity of leucine aminopeptidase in lymphotropic infections and in malignant lymphomas]. / Die Serumaktivität der Leuzinaminopeptidase bei lymphotropen Infektionen und malignen Lymphomen.

Increases of the leucine aminopeptidase according to data of literature are regarded as a sensitive parameter of lesions in liver diseases. In patients with active generalised lymphadenitis (viral infections, toxoplasmosis) as a typical enzyme constellation a relatively strong leucine aminopeptidase increase in the serum with missing or relatively slight enzyme deviation of the transaminases could be found. The quotient of the activities of LAP/ALAT was clearly above that of inflammatory liver diseases. In chronic lymphatic leukosis, plasmocytoma and malignant lymphomas the leucine aminopeptidase serum activities were within the normal. Increases of leucine aminopeptidase in lymphotropic infections are probably partly of extrahepatic origin.

Profile of intracytoplasmic lysozyme in normal tissues, myeloproliferative disorders, hairy cell leukemia, and other pathologic processes. An immunoperoxidase study of paraffin sections and smears.

Intracytoplasmic lysozyme (muramidase) may be readily identified in paraffin sections of tissues fixed in formalin or Zenker's acetic acid and in smears of peripheral blood or bone marrow using an immunoperoxidase technique. Sites of intracellular lysozyme in normal human tissues and in various specimens from patients with myeloproliferative and lymphoproliferative disorders, hairy cell leukemia, granulomatous diseases, toxoplasmic lymphadenitis, and other pathologic processes were defined by this method. Intracellular lysozyme was demonstrated in mature and immature neutrophilic and eosinophilic myeloid cells, in monocytic cells, and in some types of histiocytes and had a limited distribution in normal tissues. The neoplastic cells of hairy cell leukemia were devoid of intracytoplasmic lysozyme. Identification of intracellular lysozyme, as determined by the immunoperoxidase technique, was compared with various cytochemical methods, particularly chloroacetate esterase and alpha-naphthyl butyrate esterase studies, for detection and characterization of myeloid cells, monocytes, and histiocytes.

Histochemistry of normal and diseased human lymph nodes.

Gomori's metal precipitate technique was used to demonstrate the phosphatase activity of the human cervical lymph node in health and disease, using four different phosphate esters (sodium beta-glycerophosphate and adenosine triphosphate at pH 9, riboflavin 5'-phosphate at pH 9.2 and 5'-monophosphoric acid at pH 8.3). In fetal lymph nodes, using 5'-monophosphoric acid, an outstanding positive activity was noticed in the lymphatic follicles. With the other three substrates there was either no nodular reaction or just a narrow rim of positive activity around the follicles, the internodular tissue being negative with all four substrates used. With chronic non-specific lymphadenitis the enzyme hydrolysing the three substrates (beta-glycerophosphate, riboflavin 5'-phosphate and adenosine triphosphate) began to make their appearance. It seems that with lymphadenitis, a qualitative change of the phosphatase activity takes place. A special characteristic pattern of phosphatase activity has been described in both 'early' and 'caseating' tuberculous lymphadenitis. In malignant lymphomas it was noticed that no activity was encountered with any of the four substrates in reticulum cell sarcoma. However, in lymphosarcoma a positive activity was obtained when either beta-glycerophosphate or adenosine triphosphate substrates was used, to the extent that one can depend upon this characteristic phosphatase activity in differentiating between reticulum cell sarcoma and lymphosarcoma. However, no enzymatic activity was obtained when the other two phosphate esters were used.

[Lymphoreticular proliferations in the skin. Cytochemical and immunocytological studies in lymphadenosis benigna cutis]. / Cytochemische und immuncytologische Untersuchungen bei Lymphadenosis benigna cutis

In 8 patients with lymphadenosis benigna cutis (LABC) cytochemical and in 2 of them immunocytological studies have been performed. 1) In patients with LABC we find ectopic organoid proliferations of "lymphfollicle"-like structures within the dermis which predominately consist of small lymphocytes and large reticulum cells. Immunocytological differentiation of the lymphocytes leads to the characterization of B- and T-lymphocytes in a ration 2:1. 2) Large reticulum cells represent a peculiarly remarkable cell class in infiltrates of LABC. Because of their typical arrangement disseminated within the lymphocytic infiltrate they have been designated as "starry sky" cells. Cytochemically they are characterized by an unusual high content of nonspecific esterases and acid phosphatase, most of them show phagocytized basophilic bodies. Because of their shape, arrangement and enzymcytochemical behaviour these cells can be referred to as typical for the LABC disease. 3) Monocytes cannot be found within the "lymphfollicles". Mast cells and connective tissue cells are rarely observed. Polymorphonuclear granulocytes can be demonstrated in great numbers in any part of the involved cutis when there is an insect bite in history. 4) As a reaction of the ectopic proliferation of lymphoreticular tissue within the dermis there is an activation of the surrounding connective tissue with an increase of the alkaline phosphatase activity within these cells, new formation of collagen fibres and strong proliferation of alkaline phosphatase positive capillaries. 5) Etiopathologically it is stressed, that in LABC for example an insect bite induces stimulation of hematopoietic potentialities of undifferentiated mesenchymal germ centres within the cutis takes place, leading to the development of ectopic of "lymphfollicle" like structures.