PMID- 10070034
OWN - NLM
STAT- MEDLINE
DCOM- 19990415
LR  - 20190205
IS  - 0002-9513 (Print)
IS  - 0002-9513 (Linking)
VI  - 276
IP  - 3
DP  - 1999 Mar
TI  - Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells.
PG  - G591-8
LID - 10.1152/ajpgi.1999.276.3.G591 [doi]
AB  - CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, 
      monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer 
      cells by LPS remains controversial. The purpose of this study was to determine if 
      different methods used to isolate Kupffer cells affect CD14. Kupffer cells were 
      isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and 
      differential centrifugation using Percoll gradients and cultured for 24 h before 
      experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well 
      as from peritoneal macrophages. Western blotting showed that Kupffer cells 
      prepared with collagenase possess CD14; however, it was absent in cells obtained 
      by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared 
      with collagenase was increased transiently to levels around 300 nM by addition of 
      LPS with 5% rat serum, which contains LPS binding protein. This increase in 
      intracellular calcium was totally serum dependent. Moreover, LPS-induced 
      increases in intracellular calcium in Kupffer cells were blunted significantly 
      (40% of controls) when cells were treated with phosphatidylinositol-specific 
      phospholipase C, which cleaves CD14 from the plasma membrane. However, 
      intracellular calcium did not increase when LPS was added to cells prepared by 
      collagenase-Pronase perfusion even in the presence of serum. These cells were 
      viable, however, because ATP increased intracellular calcium to the same levels 
      as cells prepared with collagenase perfusion. Tumor necrosis factor-alpha 
      (TNF-alpha) mRNA was increased in Kupffer cells prepared with collagenase 
      perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; 
      however, serum did not increase TNF-alpha mRNA in cells isolated via 
      collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased 
      CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings 
      indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase 
      perfusion does not, providing an explanation for why Kupffer cells do not exhibit 
      a CD14-mediated pathway when prepared with procedures using Pronase. It is 
      concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when 
      prepared gently.
FAU - Ikejima, K
AU  - Ikejima K
AD  - Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, 
      University of North Carolina, Chapel Hill, North Carolina 27599-7365, USA.
FAU - Enomoto, N
AU  - Enomoto N
FAU - Seabra, V
AU  - Seabra V
FAU - Ikejima, A
AU  - Ikejima A
FAU - Brenner, D A
AU  - Brenner DA
FAU - Thurman, R G
AU  - Thurman RG
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Am J Physiol
JT  - The American journal of physiology
JID - 0370511
RN  - 0 (Lipopolysaccharide Receptors)
RN  - 0 (RNA, Messenger)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - 25249-22-3 (Poly I)
RN  - EC 3.4.24.- (Collagenases)
RN  - EC 3.4.24.- (Pronase)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Animals
MH  - Calcium/metabolism
MH  - Cell Line/drug effects/metabolism
MH  - Collagenases/pharmacology
MH  - Female
MH  - Intracellular Membranes/metabolism
MH  - Kupffer Cells/*drug effects/*metabolism
MH  - Lipopolysaccharide Receptors/*drug effects
MH  - Mice
MH  - Poly I/pharmacology
MH  - Pronase/*pharmacology
MH  - RNA, Messenger/metabolism
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Tumor Necrosis Factor-alpha/genetics
EDAT- 1999/03/10 00:00
MHDA- 1999/03/10 00:01
CRDT- 1999/03/10 00:00
PHST- 1999/03/10 00:00 [pubmed]
PHST- 1999/03/10 00:01 [medline]
PHST- 1999/03/10 00:00 [entrez]
AID - 10.1152/ajpgi.1999.276.3.G591 [doi]
PST - ppublish
SO  - Am J Physiol. 1999 Mar;276(3):G591-8. doi: 10.1152/ajpgi.1999.276.3.G591.