Presence of α1-Antichymotrypsin and α1-Antitrypsin in Haematopoietic and Lymphoid Tissue Cells as Revealed by the Immunoperoxidase Method

Path. Res. Pract. 169, 287-297 (1980) Department of Pathology, University of Athens, Greece Institute of Pathology, University of Kiel, FRG

Presence of at-Antichymotrypsin and at-Antitrypsin in Haematopoietic and Lymphoid Tissue Cells as Revealed by the Immunoperoxidase Method c.

s. PAPADIMITRIOU, H. STEIN, and N. X. PAPACHARALAMPOUS Summary

The aim of the present study was to determine the significance of at·antichymotrypsin and atantitrypsin as markers for the mono histiocytic and myeloid cell series. For this investigation, paraffin sections from tonsil, lymph node, and bone martow biopsies and cytological smears of blood and bone marrow were studied with the immunoperoxidase (PAP) method for the presence of at-antichymotrypsin and at-antitrypsin in various types of haematopoietic and lymphoid tissue cells. The results of immunostaining of paraffin sections showed that cells of the monohistiocytic cell series, including tingible body macrophages, sinus histiocytes, and epithelioid cells, are moderately or strongly positive for at-antichymotrypsin and negative for at-antitrypsin. Epithelioid cells were the only exception, since they were sometimes weakly positive for at-antitrypsin. An opposite staining pattern was observed in the myeloid cell series. Mast cells were strongly positive for both substances, whereas dendritic reticulum cells and lymphoid cells were negative. Endothelial cells stained for at-antichymotrypsin, but not for aj-antitrypsin. In formalin-acetone-fixed blood and bone marrow smears, lymphocytes, erythroblasts, and megakaryocytes were consistently negative for arantichymotrypsin and at-antitrypsin, whereas polymorphs and myelocytes were strongly positive for at-antitrypsin. ]n contrast to paraffin sections, the smears revealed ajanti chymotrypsin not only in a proportion of monocytes, but also in a variable percentage of myelocytes and polymorphs. The presented findings indicate that aj-antichymotrypsin is a highly characteristic, although not a specific, marker of cells of the monohistiocytic cell series when it is demonstrable on paraffin sections.

Introduction urAntitrypsin (Schultze et ai., 1955, 1962) and urantichymotrypsin (Heimburger and Haupt, 1965, 1966) are two protease inhibitors that neutralize the enzymatic activity of enzymes such as trypsin, chymotrypsin, and elastase. urAntitrypsin, which also shows antichymotrypsin-like properties (Venge et aI., 1975), has been studied more extensively than urantichymo20 Path. Res. Pract. Vol. 169

288 . C. S. Papadimitriou, H. Stein, and N. X. Papacharalampous

trypsin. An at-antitrypsin deficiency has been related to some pathological conditions, e.g., emphysema and certain forms of liver cirrhosis (Laurell and Eriksson, 1963; Sharp, 1971, 1976; Talamo, 1975). It is generally assumed that arantitrypsin is produced by liver cells. Evidence of production of ar antitrypsin or at-antichymotrypsin outside the liver, e.g., in haematopoietic or lymphoid tissue, has not been presented. In a recent study using the peroxidase-antiperoxidase complex (PAP) method on paraffin sections from Hodgkin's disease, our research group showed that, besides Hodgkin and Sternberg-Reed cells, cells of histiocytic nature were often positive for arantichymotrypsin, whereas polymorphs and lymphoid cells were negative (Papadimitriou et aI., 1978). This observation appeared important enough for us to investigate the use of at-antichymotrypsin as a specific marker of cells of monohistiocytic origin. If such a marker were applicable on paraffin sections, it would be invaluable to histopathologists for helping to distinguish the various types of cells of haematopoietic tissue. In the present study, myeloid cells and cells of lymphoid tissue were further investigated with the PAP method on paraffin sections and cytological smears for the possible presence of arantitrypsin and at-antichymotrypsin.

Materials and Methods Paraffin sections Ten tonsils from children up to 12 years old, 10 lymph nodes with reactive lymphadenitis, two lymph nodes with toxoplasmosis, four lymph nodes with tuberculosis, two lymph nodes with sarcoidosis, and five bone marrow biopsies from the iliac crest were used. Tonsil specimens and half of each lymph node were cut into thin (up to 3 mm) slices and fixed in buffered formalin at 4 °C for 6-8 hours. Small bone marrow pieces, free of bone fragments, were fixed in a similar manner; decalcification was not applied. The second half of each lymph node was fixed in 10% formalin at room temperature for 12-36 hours. Dehydration and embedding in paraffin were performed by the routine procedure in all instances. Cytological smears Peripheral blood smears from 20 healthy individuals and two persons with chronic myelogenous leukaemia and bone marrow smears from 10 individuals who did not have haematological malignancies were obtained. All smears were fixed in buffered formalin-acetone solution for 30 secon9s as described by Mason et a!. (1975 ). Immunoperoxidase tech~ique For the demonstration of intracellular aI-antitrypsin and al-antichymotrypsin, the peroxidaseantiperoxidase (PAP) method of Sternberger et a!. (1970) was applied on 5 Ilm paraffin sections, using the basic technique described by Taylor (1974). Endogenous peroxidase activity was blocked with methanol containing 0.3% H10 1. Anti-at-antichymotrypsin and anti-aj"antitrypsin

a,-Antichymotrypsin and at-Antitrypsin in Haematopoietic and Lymphoid Cells . 289 rabbit antisera were purchased from Behringwerke (Marburg, W. Germany). They were applied at a dilution of 1:100. Swine anti-rabbit IgG antiserum and the PAP complex were obtained from Dakopatts (Copenhagen, Denmark) and were used at dilutions of 1:40 and 1:60, respectively. Before immunostaining, the paraffin sections were treated with normal swine serum to reduce nonspecific staining. After fixation and before staining, the smears were thoroughly washed in distilled water. Substitution of non immune rabbit serum for the antisera and a slide treated with diaminobenzidine reagent alone served as controls. All preparations were counterstained with haematoxylin or Giemsa. The intensity of the reaction was estimated according to the following scale: absolutely negative (-), slightly positive (+), moderately positive (+ +), strongly positive (+++), very strongly positive (++++).

Enzyme cytochemical staining A series of cytological smears were fixed in calcium-formalin and stained for nonspecific anaphthyl acetate esterase according to the method of Braunstein (1959) . Paraffin sections adjacent to those used for PAP immunostaining were cut from all tonsils, lymph nodes, and bone marrow biopsies and stained for naphthol AS-D-chloroacetate esterase according to the method of Leder (1964).

Results Paraffin sections The results of immunostaining of the various types of cells for al-antitrypsin and al-antichymotrypsin on paraffin sections are given in Table 1. Lymphoid cells, including lymphocytes in Band T regions, germinal centre cells, and immunoblasts, were negative for both al-antitrypsin and al-antichymotrypsin in all cases studied (Figs. 1 and 2). Table 1. a,-Antichymotrypsin and at-antitrypsin antigenicity in various cells on paraffin sections Type of cell Lymphoid cells Polymorphs Mast cells Tingible body macrophages Dendritic reticulum cells Endothelial cells Sinus histiocytes Epithelioid cells Myelocytes Erythroblasts Megakaryocytes

- = negative, + = slightly positive, + + + + + + = very strongly positive

a,-Antichymotrypsin

at-Antitrypsin

-to+ ++++ ++ to ++++

++++ ++++

+++ ++++ +++

- to +

- to

++++

+

= moderately positive,

+++

= strongly

positive,

Fig. 1. Immunostaining (PAP method) of a reactive germinal centre for al-antichymotrypsin. There are many positive macrophages in the germinal centre. X 125.

Fig. 2. Immunostaining (PAP method) of a lymph node section displaying follicular hyperplasia for aI-antitrypsin. Macrophages are negative, while mast cells in the interfollicular area are strongly positive. x 125.

Fig. 3. Immunostaining (PAP method) of a lymph node section for urantitrypsin. Polymorphs present in vessels are strongly positive, while other cells, including endothelial cells, are negative. x 160.

Fig. 4. Immunostaining (PAP method) of a lymph node section for urantichymotrypsin. Endothelial cells are strongly positive. x 160.

292 . C. S. Papadimitriou, H. Stein, and N. X. Papacharalampous

Fig. 5. Immunostaining (PAP method) of a l ymph node section for uJ-antichymotrypsin. Histiocytes in a sinus are strongly positive. x 312.

Fig. 6. Immunostaining (PAP method) of a section from a lymph node with tuberculosis for antichymotrypsin. Epithelioid cells and Langhans' giant cells are strongly positive. X 125.

U 1-


Polymorphs were strongly positive for ut-antitrypsin in all cases and usually negative for urantichymotrypsin (Figs. 3 and 4). In specimens that had been fixed for a short time, however, some polymorphs were slightly positive and a few were moderately positive for urantichymotrypsin. Mast cells (Fig. 2) were strongly positive for both substances in all cases. The true nature of such cells was easily recognized after staining of adjacent sections with Giemsa or for naphthol AS-D-chloroacetate esterase. In sections stained for urantichymotrypsin, some cells lying in extrafollicular areas showed a positive reaction resembling that of the mast cells. These cells did not contain metachromatic granules and were negative for naphthol AS-Dchloroacetate esterase; they were considered to be of monohistiocytic nature. Germinal centre macrophages with intracytoplasmic tingible bodies were moderately to strongly positive for urantichymotrypsin (Fig. 1). The intensity of the immunostaining appeared to correspond to the phagocytic activity of the cells. In contrast, macrophages were always negative for urantitrypsin (Fig. 2). We were unable to detect any cells with the morphology and localization of so-called dendritic reticulum cells that were positive for either of the studied substances. A strongly positive reaction for urantichymotrypsin and a negative reaction for urantitrypsin were also demonstrated in endothelial cells of capillaries and small vessels and in histiocytes filling lymph node sinuses (Figs. 3,4, and 5).

Epithelioid cells in all cases of tuberculosis, sarcoidosis, and toxoplasmosis were strongly positive for urantichymotrypsin (Fig. 6), whereas they were usually negative, or only slightly positive for ut-antitrypsin. In bone marrow sections, myelocytes were strongly positive for urantitrypsin and slightly or moderately positive for ut-antichymotrypsin. Erythroblasts and megakaryocytes showed a negative reaction for both substances. Table 2.

Lymphocytes Polymorphs Monocytes Myelocytes Erythroblasts Megakaryocytes

- = negative, + = slightly

+ +++

positive, == very strongly positive

- to -

to

- to

++

++ ++ ++

== moderately positive,


++++ +++

+++ =

strongly positive,

294 . C. S. Papadimitriou, H. Stein, and N. X. Papacharalampous

Blood and bone marrow smears (Table 2).

In all cases, lymphocytes were absolutely negative for both urantichymotrypsin and urantitrypsin. Polymorphs were always strongly positive for Ur antitrypsin (Fig. 7), whereas they displayed variable stainability for urantichymotrypsin. A number of polymorphs, varying from person to person, showed slight or moderate staining for urantichymotrypsin (Fig. 8) in all but one case. In the latter case, they were strongly positive. The number of monocytes in each case was estimated on morphological grounds and after staining for nonspecific esterase. After immunostaining, only a small number of monocytes were moderately positive for u1-antichymotrypsin (Fig. 8). By comparing the cytological smears stained for urantichymotrypsin with those stained for nonspecific esterase, we found that Ur antichymotrypsin-positive cells made up only 10% of the esterase-positive cells. Myelocytes showed a strongly positive reaction for urantitrypsin (Fig. 9) and variable stainabilitiy for urantichymotrypsin, comparable to that of polymorphs. Erythroblasts and megakaryocytes were negative for both substances.

Fig. 7. Immunostaining (PAP method) of a peripheral blood smear for aI-antitrypsin. Polymorphs are strongly positive, while lymphocytes are negative. X 640.

at-Antichymotrypsin and ai-Antitrypsin in Haematopoietic and Lymphoid Cells . 295 ,".

Fig. 8. Immunostaining (PAP method) of a peripheral blood smear for al-antichymotrypsin. Four polymorphs and one monocyte show moderate positivity. x 500 .

••

• Fig. 9. Immunostaining (PAP method) of a peripheral blood smear from a patient with chronic myelocytic leukaemia for acantitrypsin. Myelocytes are strongly positive. x 500.

296 . C. S. Papadimitriou, H. Stein, and N. X. Papacharalampous

Discussion The results of this study showed that cells of myelocytic or monohistiocytic origin are positive for arantitrypsin and/or arantichymotrypsin. The presence of these two substances in such cells might be of functional significance. It has been well established that, in collaboration with armacroglobulin, ar antitrypsin and arantichymotrypsin play an important role in neutralizing the chymotrypsin-like enzymes produced by leucocytes (Venge et al., 1975). The assumption that the presence of at-antitrypsin and arantichymotrypsin in leucocytes is related to the functional state of the cells is supported by our finding that the intensity of immunostaining for at-antichymotrypsin in macrophages corresponded to their phagocytic activity. We also demonstrated that monohistiocytic and myelocytic derivatives differ in their contents of arantichymotrypsin and arantitrypsin. As a rule, monohistiocytic cells on paraffin sections were strongly positive for arantichymotrypsin and negative for arantitrypsin. Epithelioid cells were the only exception, being slightly positive for arantitrypsin. In contrast, myelocytes and polymorphs were strongly positive for arantitrypsin and negative to moderately positive for at-antichymotrypsin. The observed discrepancy in stainability of polymorphs for arantitrypsin and at-antichymotrypsin between paraffin sections and smears is difficult to explain. It might be an artefact caused by fixation and tissue processing. The finding that only a very small proportion of monocytes in peripheral blood were positive for arantichymotrypsin could be attributed to their functional state or functional immaturity. The results of this study show that a strongly positive reaction for ar antichymotrypsin and a negative or slightly positive reaction for arantitrypsin in cells, especially on paraffin sections, are evidence in favour of their monohistiocytic origin, whereas the opposite staining pattern favours a myelocytic origin. Since lymphoid cells are absolutely negative and mast cells are strongly positive for both arantichymotrypsin and ai-antitrypsin, the detection of these substances by means of PAP immunostaining of paraffin sections appears to be a reliable criterion for the characterisation of cells in reactive and neoplastic conditions. Other enzymes that can be easily detected in paraffin sections are lysozyme, which is also demonstrable with the PAP method (Mason and Taylor, 1975), and naphthol AS-D-chloroacetate esterase, which is demonstrable with an enzyme histochemical method (Leder, 1964). Since the cells involved in reactive and, particularly, in neoplastic conditions may lose some of their markers and functional properties, it is important to have several methods available for cell characterisation.

at-Antichymotcypsin and at-Antitrypsin in Haematopoietic and Lymphoid Cells . 297

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Received September 5, 1979 . Accepted October 15, 1979

Key words: arAntichymotrypsin - arAntitrypsin - Lymphoid cells - Monohistiocytic cells

Prof. Dr. H. Stein, Institut fiir Pathologie, Klinikum der Universitiit Kiel, Hospitalstr. 42, D-2300 Kiel 1, FRG