Lysosomal and Ultrastructural Changes in Human "Toxic" Neutrophils During Bacterial Infection

Abstract

"Toxic" neutrophils from humans with severe bacterial infections, identified by the presence of Döhle bodies, "toxic" granules, and vacuoles were shown to differ from normal neutrophils both in ultrastructure and in lysosome activity. Döhle bodies were identified as lamellar aggregates of rough endoplasmic reticulum. Toxic granules corresponded to the azurophilic granules usually identified by Romanowsky stains only in neutrophil precursors. By electron microscopy such granules were large, electron-dense, and peroxidase positive; they could usually be distinguished from the smaller, less dense, "specific" granules also present in control neutrophils, but in the latter they became visible by light microscopy only after prolonged staining or following fixation with glutaraldehyde. These observations suggest that toxic granules represent an abnormal staining reaction of the large dense granules in the toxic cells, and not phagocytized material, newly formed abnormal granules or autophagic bodies. Alkaline phosphatase activity was significantly greater in toxic neutrophils than in normal ones; 80% of the activity of both was located in the lysosome fraction. Beta glucuronidase was normal. Total acid phosphatase was normal, but the percentage located in the nonlysosome fraction of toxic neutrophils was increased, suggesting that lysosomes were "labilized." Formation of neutral red vacuoles in supravitally stained preparations, an index of lysosome activity, occurred more rapidly in toxic neutrophils. This reaction paralleled degranulation and the formation of clear vacuoles in unstained wet mounts and could be blocked by colchicine, a lysosome stabilizer, or enhanced by procedures which activate lysosomes. "Autophagic" vacuoles were observed by electron microscopy in some toxic neutrophils. These observations are discussed in relation to the concept that the "toxic" neutrophils in severe bacterial infection reflect cellular immaturity and/or stimulation or degeneration.

Enhanced Phagocytic Capacity THE BIOLOGIC BASIS FOR THE ELEVATED HISTOCHEMICAL NITROBLUE TETRAZOLIUM REACTION

“…The pathogenesis of alterations causing toxic granulation and vacuolization of PMNs is unknown, but data have suggested that there are changes in the cell membrane (11). These PMNs become more permeable to Romanovsky stains after which the azurophil granules stain deeply as "toxic granulation."…”

“…These PMNs become more permeable to Romanovsky stains after which the azurophil granules stain deeply as "toxic granulation." The Ddhle bodies frequently seen in such cells represent an aggregate of rough endoplasmic reticulum, and the vacuolization of these neutrophils probably results from formation of secondary lysosomes which occur as phagolysosomes or autophagic vacuoles (11). The presence of toxic granulation correlates with an elevated histochemical NBT reaction (7).…”

“…The NBT histochemical test and the presence of toxic granulation and vacuolization of PMNs which may occur during acute bacterial infection have both been utilized for this purpose (1,11). Unfortunately, neither is totally specific nor altogether sensitive.…”

The Interaction Between Human Monocytes and Red Cells

“…Glutaraidehyde-fixed, washed suspensions of cells were incubated in the diaminobenzidine H202 medium of Graham and Karnovsky (18) for 15-30 rain, washed, and for electron microscopy, postfixed in osmium as described above. By this method the large "azurophilic" granules of neutrophils were strongly stained (19), whereas the monocytes were either unstained or exhibited fine stippling by light microscopy and staining of smaller granules by electron microscopy (see below). in inmmnohemolytic anemias in man, induced red cell binding; IgM antibodies produced complement-dependent mixed agglutination and phagocytosis.…”

Inquiry into the causes and significance of cytoplasmic vacuolation of neutrophils in the peripheral circulation

“…Included in this group were the only two cases of septicemia due to enteric bacteria. McCall <1969 and1979> and Bigley <personal communication) claim that the vacuolation was due to the presence of bacterial products and not the bacteria themselves so I felt it was not necessary to expose the mice to live bacteria at this time. Aliquots of the boiled suspension were plated on nutrient agar to assure that the bacteria were killed.…”