Endotoxin infusion primes elicited neutrophils and Kupffer cells for platelet-activating factor-induced and tripeptide formyl-methionine-leucine-phenylalanine-induced basal free intracellular calcium concentration responses.
OBJECTIVE
To determine whether in vivo infusions of bacterial endotoxin prime rat Kupffer cells and elicited neutrophils by altering the basal free intracellular calcium concentration or the response of free intracellular calcium to platelet-activating factor or the tripeptide formyl-methionine-leucine-phenylalanine (f-met-leu-phe).


METHODS
Prospective, randomized, controlled study.


METHODS
Laboratory of a university medical school.


METHODS
Adult male Sprague-Dawley rats.


METHODS
Rats were infused with sterile saline or with Escherichia coli endotoxin for 3 or 30 hrs via a venous catheter or an osmotic minipump. Rats were anaesthetized, and the livers were perfused in situ with a collagenase-containing buffer. Kupffer cells and elicited neutrophils were isolated by centrifugal elutriation, and were partially purified over Ficoll gradients.


RESULTS
Basal free intracellular calcium concentrations were determined with the calcium indicator, fluo-3. Various concentrations of platelet-activating factor or f-met-leu-phe were then added, and any alterations in free intracellular calcium values were quantified. Neither acute (3-hr) nor chronic (30-hr) infusions of endotoxin altered basal free intracellular calcium values. F-met-leu-phe-induced increases in free intracellular calcium concentrations were positively correlated with the percentage of neutrophils in Kupffer cell fractions. Three-hour infusions of endotoxin, which caused a significant inclusion of neutrophils in the Kupffer cell fraction, resulted in an enhanced response to f-met-leu-phe. This response was considered to be primarily due to the presence of neutrophils, because preparations that were > 95% Kupffer cells did not respond to f-met-leu-phe. Platelet-activating factor-induced increases in free intracellular calcium concentrations were also positively correlated with the percentage of neutrophils. However, Kupffer cells isolated from rats infused for 30 hrs with endotoxin contained < 12% neutrophils, and exhibited a ten-fold greater response to platelet-activating factor than did Kupffer cells isolated from saline-infused rats.


CONCLUSIONS
In vivo infusions of endotoxin result in an enhanced response to platelet-activating factor by rat Kupffer cells. F-met-leu-phe does not appear to induce an increase in free intracellular calcium values in rat Kupffer cells, but it appears to do so in endotoxin-elicited neutrophils. These observations indicate that changes in calcium homeostasis may be one mechanism by which endotoxin primes Kupffer cells and elicited neutrophils for enhanced production of superoxide anion and/or nitric oxide.
Hagar
A F
AF
Department of Physiology, Louisiana State University Medical Center, New Orleans 70112.
duSapin
K
K
Spitzer
J A
JA
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002469:Cell Separation; D004731:Endotoxins / Q000494:pharmacology*; D004926:Escherichia coli; D005434:Flow Cytometry; D007728:Kupffer Cells / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology*; D011446:Prospective Studies; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley
D004731:Endotoxins; D010972:Platelet Activating Factor; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002118:Calcium