Suppression of local and systemic responses in streptococcal cell wall-induced acute inflammation of the air pouch by cyclosporine A. Comparison with the effects of two anti-inflammatory bis-benzimidazoles.

Injection of streptococcus group A cell wall-derived peptidoglycan polysaccharide into a subcutaneous air pouch causes local outpouring of neutrophils and macrophages and distant hemopoietic proliferation in spleen and bone marrow. Cyclosporine A (CyA) suppressed neutrophil accumulation and all cell lines of hemopoiesis. trans-1,2-Bis(5-amidino-2-benzimidazolyl)ethene (BBE) also interfered with neutrophil exudation, yet reduced only the erythroid component of the hemopoietic process. The ethane analogue of BBE, on the other hand, did not prevent neutrophil emigration, but held down splenic erythropoiesis and myelopoiesis. All three compounds stimulated streptococcus group A cell wall-derived peptidoglycan polysaccharide uptake by pouch macrophages. CyA being the least active, BBE and its ethane analogue also produced a shift of wear-and-tear pigment from large numbers of small splenic macro-phages into small numbers of large macrophages. The pouch model is very useful in the study of anti-inflammatory compounds and has furnished the first evidence of CyA interference with massive neutrophilic infiltration and with hemopoietic signals.     

Identification of a subpopulation of primary granules in human neutrophils based upon maturation and distribution. Study by transmission electron microscopy cytochemistry and high voltage electron microscopy of whole cell preparations

Two main classes of primary granules have been identified in human neutrophils by peroxidase cytochemistry and electron microscopy, spherical granules and elongated granules, containing crystals with well-defined periodicity. In the present study, we report another subpopulation of primary granules, distinguishable from the other primary granules by their small size, strong peroxidase reactivity under selective incubation conditions with diaminobenzidine, stage of neutrophil maturation at which they appear, and distinctive morphological characteristics. Early promyelocytes, reacted with diaminobenzidine before fixation, demonstrate no reactivity in the large primary granules, but strong peroxidase activity in the endoplasmic reticulum and Golgi apparatus. At the end of the promyelocyte stage of maturation, when the endoplasmic reticulum decreases in peroxidase reactivity, small, round or elongated granules (100 to 200 nm) are observed, reacting strongly for peroxidase. These small granules persist during maturation. The data suggest that these small granules are packaged after the large primary granules and before the secondary granules. All three classes of primary granules exhibit peroxidase activity when fixed prior to incubation in diaminobenzidine at neutral or alkaline pH. Distinctive morphological characteristics of the small primary granules are observed in circulating neutrophils. The small granules are arranged in chains or clusters primarily at the cell margin or uropod in moving cells and are aligned along the axis of cell polarity. This granule association is more evident in adherent neutrophils, particularly after short incubation with phorbol myristate acetate. Chains consisting of as many as 17 small granules are observed. Stereo high voltage electron microscopy of whole-mount preparations of adherent neutrophils reveals chains of small granules apparently interconnected by microtrabeculae. Thus, the small granules observed in thin sections do not represent a transverse section of elongated granules, and the interconnection of granules by microtrabeculae may determine the arrangement of granules. These data support the existence of subpopulations of primary granules which contain distinct forms of myeloperoxidase.     

Suppression of streptococcal cell wall-induced arthritis by a potent protease inhibitor, bis(5-amidino-2-benzimidazolyl)methane

Bis(5-amidino-2-benzimidazolyl)methane, a powerful synthetic trypsin inhibitor, proved to be highly effective in suppressing the arthritis induced by streptococcal cell wall fragments in Lewis rats. It reduced not only the degree of synovitis, osteitis, and hematopoietic hyperplasia in the distal extremities, but also the degree of associated granulomatous inflammation in the liver. The results suggest that trypsin-like proteases play an important role in this arthritis model and that inhibitors may be useful in the treatment of similar arthritic conditions in humans.     

Opsonized streptococcal cell walls cross-link human leukocytes and erythrocytes by complement receptors.

Serum-opsonized group A streptococcal cell walls, consisting of peptidoglycan-polysaccharide polymers (PG-APS), induced monolayers of human neutrophils, monocytes, and eosinophils to aggregate. When erythrocytes were present in the incubation medium, they also were associated with the leukocyte aggregates. By immunofluorescence staining, PG-APS was localized at the site of cell-to-cell contact. By scanning electron microscopy the cells appeared to adhere to one another by surface contact; filopodia often acted as connectors, particularly in leukocyte-erythrocyte interaction. Cellular binding of PG-APS and aggregation were dependent upon C3 fixation. No aggregation was observed when heat-inactivated serum was used as an opsonin. In contrast to peptidoglycan, an activator of the alternative complement pathway, the group-specific polysaccharide moiety of PG-APS induced no cellular aggregation. Rosette formation was observed in suspensions when neutrophils were incubated with erythrocytes coated with C3b-opsonized PG-APS. Cell monolayers bound serum-opsonized PG-APS, but aggregation was observed only when serum was present in the incubation medium. Similar results were obtained with C5-deficient serum. No aggregation was observed with heat-inactivated serum or bovine serum albumin. A heat-labile serum component(s) appears to be required to elicit leukocyte aggregation. It is suggested that C3 fixed to PG-APS acts as a bridge to link cells together in clusters as a result of common recognition of C3 by leukocyte and erythrocyte complement receptors.     

Microtubule organization of unstimulated and stimulated adherent human neutrophils in Chediak-Higashi syndrome

The numbers and length of centriole-associated microtubules of two patients with Chediak-Higashi syndrome (CHS) were examined. Detergent- extracted whole-mount preparations of adherent cells were studied by stereo high-voltage electron microscopy. Under conditions of random migration, neutrophils from both patients had a microtubule organization similar to that of the control; microtubule numbers (28 +/- 3) and length (7.0 +/- 2.8 micron) were within normal range. When cells were treated with phorbol myristate acetate (PMA), differences in the response of the two patients were noted. Neutrophils from patient No. 2 and the control showed a significant rise in numbers (38 +/- 5) and length (9.5 +/- 3.6 micron) of microtubules. In contrast, neutrophils from patient No. 1 were unresponsive to PMA treatment. Because vitamin C is used therapeutically in CHS patients and has been shown to correct microtubule-related cell function, neutrophils were exposed to ascorbic acid. A significant increase in microtubule numbers (35 +/- 6) was observed in cells from the control and patient No. 2 after ascorbate treatment; neutrophils from patient No. 1 showed no increase in microtubule numbers. While ascorbic acid did not affect microtubule length in the control cells, it caused a significant increase in microtubule length in neutrophils from both patients. Results suggest that adherent CHS neutrophils contain centriole-associated microtubules which are normal in number and length. However, differences between patients are observed regarding neutrophil responsiveness to stimuli which induce microtubule polymerization.     

Compartmentalization of cyclic GMP-dependent protein kinase in formyl- peptide stimulated neutrophils

The presence and physiologic role of cyclic GMP-dependent protein kinase (G-kinase) in human neutrophils was investigated by Western blot analysis and immunocytochemistry. Small quantities of G-kinase were found in the cytoskeletal-enriched fraction of neutrophil lysates as detected by Western blots using a polyclonal antibody raised against bovine aorta G-kinase. Immunofluorescence microscopy demonstrated in adherent neutrophils that G-kinase was localized diffusely within the cytoplasm, at the microtubule organizing center, and in the euchromatin of the nucleus. Because cyclic GMP is implicated as a modulator of neutrophil chemotaxis, G-kinase localization was investigated in neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). fMLP stimulated transient focal changes in G-kinase localization that coincided with transient changes in cell shape. G- kinase translocated over a period of 5 minutes from diffuse staining of the cytosol to filaments within the uropod of polarized cells (1 minute), to bundles of filaments associated with loss of cell polarity (2.5 minutes), and finally to more intense staining of the nuclear euchromatin (5 minutes). Optical sectioning of neutrophils by confocal laser scanning microscopy confirmed that G-kinase was restricted to specific sub-cellular compartments during cell activation. This transient localization of G-kinase was disrupted by cytoskeletal inhibitors and was augmented by 8-Br-cyclic GMP. These data provide evidence for the first time that G-kinase plays a physiologic role in human neutrophils, and support the concept of compartmentalization of cyclic nucleotides during neutrophil activation.     

Transient granulocyte maturation arrest: discovery by flow cytometry of a variant form of agranulocytosis

A 5-year-old child presented with pneumonia and agranulocytosis. A Wright-stained peripheral blood smear showed only cells which had the morphological appearance of lymphocytes, plus a few monocytes and eosinophils. A bone marrow aspirate smear showed a complete lack of recognizable granulocyte precursors. However, the admission CBC and differential performed by automated flow cytochemistry (Technicon Instruments Corporation H-6000) measured 32% granulocytes as determined by peroxidase activity. Cytochemical stains on the blood and marrow smears revealed that many of the cells that had the morphological appearance of lymphocytes were positive for myeloperoxidase activity. Special studies on these cells revealed them to be abnormal, intermediate granulocytes with azurophilic, peroxidase-containing primary granules, but with few secondary granules and limited lactoferrin activity. Over 28 days the child recovered, first with granulocytic hyperplasia in the marrow and then a return of the peripheral blood to normal. This is the first case report of an episode of transient "agranulocytosis" which in reality was a maturation arrest.     

Sequential events in the pathogenesis of streptococcal cell wall-induced arthritis and their modulation by bis(5-amidino-2-benzimidazolyl)methane (BABIM).

This report builds on the authors' earlier discovery of bis(5-amidino-2-benzimidazolyl)methane (BABIM) as a strong suppressive agent for streptococcal cell wall fragment-induced arthritis in the Lewis rat. As a synthetic inhibitor of trypsinlike proteases, BABIM opens up a new route to the control of inflammatory joint disease. To gain a deeper insight into the function of the compound, the authors have now studied its influence on the sequential development of the joint changes and the associated lesions in spleen and liver. Bis(5-amidino-2-benzimidazolyl)methane is shown to block acute synovitis, to retard and reduce granuloma formation in spleen and liver, to decrease neutrophilic leukocytosis, and to diminish hemopoietic hyperplasia in the bone, and thus also to mitigate the distinctive osteoclastic and chondroclastic events. The compound does not interfere with the splenic immune response, the temporary rise in hepatocytic mitotic activity, or the organ deposition of streptococcal cell walls.