Increased serum enzyme levels associated with kupffer cell reduction with no signs of hepatic or skeletal muscle injury

Abstract

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that is responsible for the survival and proliferation of monocytes and the differentiation of monocytes into macrophages, including Kupffer cells (KCs) in the liver. KCs play an important role in the clearance of several serum enzymes, including aspartate aminotransferase and creatine kinase, that are typically elevated as a result of liver or skeletal muscle injury. We used three distinct animal models to investigate the hypothesis that increases in the levels of serum enzymes can be the result of decreases in KCs in the apparent absence of hepatic or skeletal muscle injury. Specifically, neutralizing M-CSF activity via a novel human monoclonal antibody reduced the CD14(+)CD16(+) monocyte population, depleted KCs, and increased aspartate aminotransferase and creatine kinase serum enzyme levels in cynomolgus macaques. In addition, the treatment of rats with clodronate liposomes depleted KCs and led to increased serum enzyme levels, again without evidence of tissue injury. Finally, in the osteopetrotic (Csf1(op)/Csf1(op)) mice lacking functional M-CSF and having reduced levels of KCs, the levels of serum enzymes are higher than in wild-type littermates. Together, these findings support a mechanism for increases in serum enzyme levels through M-CSF regulation of tissue macrophage homeostasis without concomitant histopathological changes in either the hepatic or skeletal system.

Figure 1

Nonhuman primate (cynomolgus macaque) liver section CD163 and KCs by laser-scanning cytometery (LSC). A: Liver section from a control monkey showing many scattered KCs. B: Liver section from a drug-treated monkey (the drug is a novel neutralizing human M-CSF monoclonal antibody) showing significantly fewer KCs. C: LSC quantitation. LLL indicates left lateral lobe; and RLL, right lateral lobe. There was a significant decrease in KCs in treated animals. *P < 0.05, **P < 0.01.

Figure 2

Clinical pathology serum enzyme levels in nonhuman primates (cynomolgus macaques) after treatment with a novel neutralizing human M-CSF monoclonal antibody: AST (A), ALT (B), CK (C), and myoglobin (D). A: There were significant increases in AST mean values after treatment with a novel neutralizing human M-CSF monoclonal antibody. B and D: ALT and myoglobin mean values, respectively, did not change. C: CK values show large variability. **P ≤ 0.05, ***P ≤ 0.01 versus control. Triangles indicate control; squares indicate treated, dashed line indicates lower limit; and dotted line indicates upper limit.

Figure 3

IHC of ED2 macrophage marker on liver sections obtained from rats that are either treated with PBS (A) or CLO liposomes (B) at 24 hours after treatment. There was a marked reduction in ED2⁺ cells in liver (arrow) from CLO-treated rats. Original magnification, ×20. Image analysis of ED2 ratios (percentage of positive stained cells/total nuclei) for animals who underwent necropsy at 24, 72, or 192 hours after CLO administration (C). There was a marked reduction in ED2⁺ cells at 24 and 72 hours and a moderate reduction at 192 hours. ***P < 0.0005 versus control.

Figure 4

Clinical pathology serum enzyme levels in rats after treatment with CLO liposomes: AST (A), ALT (B), CK (C), LDH (D), and GLDH (E). There were significant increases in all enzyme values after CLO treatment within the first 48 hours. *P ≤ 0.05, **P ≤ 0.01 versus control. Triangles indicate control; squares indicate treated; dashed line indicates lower limit; and dotted line indicates upper limit.