Monocyte and Neutrophil Chemotactic Activity of Normal and Diluted Human Serum and Plasma

Abstract

The chemotactic activity of normal and diluted human serum and plasma for human monocytes and neutrophils was investigated. Undiluted, neither serum nor plasma attract monocytes, but this is attributable to an inhibitor masking chemotactic activity. When diluted 3- to 100-fold, appreciable activity for monocyte is detected. In contrast, chemotactic activity for neutrophils is already demonstrable in undiluted normal serum. Chemotactic activities are heat-labile, behave as pseudoglobulins, do not pass through a Diaflo PM 30 membrane and are not inactivated by treatment with di-isopropylfluorophosphate. They seem to be unrelated to the recognized components (or split products) of complement, kallikrein formation, clotting, fibrinolysis or isoantibody-blood cell-complement interaction. Upon complement activation of serum, neutrophil and monocyte chemotactic activities were generated which differed in several respects from the normal serum activities. The relative increase in activity upon activation of serum compared with normal serum was generally high for neutrophils but low for monocytes.

Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils [published erratum appears in J Cell Biol 1990 Mar;110(3):861]

“…Nevertheless, serum-stimulated chemokinesis comparable with the 25 % observed in response to serum would be expected since this does not depend upon the activation of complement (Jungi, 1977). However, no migration whatsoever was observed.…”

“…Chemokinesis stimulated by serum alone can be studied in these chemotaxis chambers by simply omitting the step of coating the erythrocytes with antibody. Unopsonized sheep erythrocytes do not activate the human complement cascade to any significant extent (Whaley, 1987), and the neutrophils migrate randomly on the surface in response to the chemoattractant present in human serum in which the complement cascade has not been activated (Jungi, 1977).…”

“…For measurement of chemokinesis, a similar dilution of serum was added to neutrophils plated onto a chemotaxis chamber that had not been treated with immunoglobulins. The ability of human serum in which the complement cascade has not been activated to act as a chemoattractant has been characterized (Jungi, 1977). Experiments in the absence of extracellular Ca 2+ were performed using serum diluted into Ca2+-free incubation medium containing 5 mM EGTA.…”

Dissociation of Chemotactic and Inflammatory Leukocyte Responses

“…After standing 40 min at room temperature, the red cells had sedimented and the leukocyte-rich plasma was aspirated and collected by centrifugation at 180 g for 10 min [Jungi, 1977]. Remaining erythrocytes were lysed by a 3-min exposure to buffered ammonium chloride after which the residual cells were washed, resuspended in medium 199 containing 1% BSA, and adjusted to 2 x l0 6 PMNs per milliliter.…”

Chemotaxis of Human Polymorphonuclear Leukocytes under Agarose: Lack of Requirement for Media Protein and Differential Effects of Buffer and Agarose Type on Locomotion

“…The process of heat inactivation routinely employed in preparing PHS may itself generate substances which stimulate PMN locomotion (29). It is also known that fresh human serum contains chemotactic and chemokinetic factors (30,31), and further, that exposure of untreated serum to agarose can activate the comple-, ment system (8). Although the latter properties of serum are reportedly heat sensitive, there may be sufficient variation in lability among some donor sera to leave detectable residual activity.…”