Immune modulator studies in primates: the utility of flow cytometry and immunohistochemistry in the identification and characterization of immunotoxicity.

Exposure to natural environmental products, biopharmaceuticals, or investigational adjuvants has the potential to negatively impact the immune system, resulting in either up- or downregulation of immune function (immunomodulation). Many current protocols for primate toxicologic testing call for the evaluation of changes in immune cell number (peripheral blood or tissue), alterations in the weights of immune system organs (lymph nodes, spleen, thymus), and/or increases in the overall incidence of infections or neoplasms; these data are relied upon to suggest altered immune function. However, these are informative only when clear differences in frequency and/or severity of effects can be distinguished across control and dosed groups. In the absence of such distinct morphologic or clinical pathologic changes, the identification of potential immunomodulatory effects can present a much greater challenge. Additional evaluations may be needed to detect altered immune system integrity; these are based on in vivo assessments in primates of cellular or humoral responsiveness. Immunomodulatory effects can be characterized by in vitro or in vivo immune function tests: these tests require prestudy planning to integrate assessments into ongoing toxicology programs. These methods also involve specialized training and equipment, particularly if the intent is to evaluate parameters in a GLP laboratory setting. In primate toxicology, the added costs required to perform a complete functional analysis of the immune system can be substantial, but may be warranted depending on the clinical development plans. Two analytical methods that are easily incorporated into the standard toxicology profile in primates are flow cytometry and immunohistochemistry. Flow cytometry (FC) is used to assess changes in the relative distribution of immune cell marker expression, and where marker expression is known to fluctuate with the state of cell activation, can also provide information on functional attributes of immune cells. Immunohistochemistry (IHC) provides a means to evaluate similar characteristics of immune cells within tissue sections. Used together, FC and IHC can aid in the identification of changes in immune system that may not be apparent by traditional testing procedures (such as H&E staining), thus aiding in the characterization of immune system alterations. This presentation focused on the utility of flow cytometry and immunohistochemistry in a standard primate toxicology evaluation, with representative examples showing the benefits of these technologies in the diagnosis of potential immunomodulatory effects.

The response of pulmonary vascular endothelial cells to monocrotaline pyrrole: cell proliferation and DNA synthesis in vitro and in vivo.

Monocrotaline pyrrole (MCTP) causes pulmonary vascular endothelial cell (EC) injury followed by progressive pulmonary vascular leak in vivo and the inhibition of EC proliferation in vitro. It was hypothesized that MCTP inhibits cell proliferation in vitro by interfering with cell cycle progression in a cycle phase-specific manner. Furthermore, it was proposed that early alterations in MCTP-induced lung injury leading to hypertension were associated with a similar inhibition of EC proliferation. Subconfluent cultures of bovine pulmonary artery endothelial cells (BECs) were synchronized with aphidicolin (APH), a reversible G1-S phase inhibitor. Upon removal of APH, BECs were exposed to MCTP (5 micrograms/ml) or its vehicle for a 4-h interval corresponding to either the G1-S, S-G2, or G2 through mitosis (M) phases of the cell cycle. Fluorescence-activated cell sorting (FACS) was used to identify MCTP-induced changes in cell cycle progression in BECs, and the transit of S phase cells through the cycle was characterized through the incorporation of bromodeoxyuridine (BrdU). Synchronized BECs exposed to MCTP between mid-S-G2 or G2 through M were briefly delayed in G2-M at 12 h but underwent cell division by 24 h. By contrast, BECs treated with MCTP immediately after release from APH block became arrested in G2-M at 24 h and showed evidence of continued DNA synthesis and hypertetraploidy, but they did not divide. In vivo, MCTP (3.5 mg/kg i.v.) administration caused an increase in arterial EC BrdU incorporation between Days 3 and 7, but no increase in EC density. During this same interval, pulmonary vascular permeability increased and persisted. In summary, MCTP inhibits cell proliferation in a cell cycle phase-dependent manner in vitro. The results suggest that a similar mechanism could occur in vivo and may be associated with delayed EC repair, a process that could contribute to persistent pulmonary vascular leak.

Hypertrophy and prolonged DNA synthesis in smooth muscle cells characterize pulmonary arterial wall thickening after monocrotaline pyrrole administration to rats.

Monocrotaline pyrrole (MCTP) is a highly reactive pneumotoxic metabolite of the pyrrolizidine alkaloid plant toxin monocrotaline. When administered to rats, it causes a delayed and progressive lung injury, vascular remodeling, and pulmonary hypertension. Structural remodeling consists of endothelial cell swelling followed by increased thickness of the vascular media in small pulmonary arteries and muscularization of normally nonmuscular arteries. Experiments were performed to characterize DNA synthesis and cell proliferation in vascular smooth muscle cells (VSMCs) after MCTP and to determine their relationship to changes in the thickness of the arterial medial layer of pulmonary resistance vessels. Male Sprague-Dawley rats were treated with MCTP (3.5 mg/kg, intravenously) or its vehicle (dimethylformamide). To label cells actively synthesizing DNA, rats were given the thymidine analog, bromodeoxyuridine (BrdU), 3 times by intraperitoneal injection during the 24 hr preceding euthanasia. Using immunohistochemistry, BrdU incorporation was quantified as a ratio of labeled nuclei to total nuclei. Within 5 days after MCTP administration, the thickness of the medial smooth muscle layer in arteries 60-250 microm in diameter was increased, prior to evidence of right heart hypertrophy. BrdU incorporation by VSMCs in pulmonary arteries was not different in vehicle- and MCTP-treated rats for the first 48 hr after treatment. However, MCTP caused a significant increase in DNA synthesis in VSMC on days 3-8 in arteries up to 250 microm in diameter. Although increased DNA synthesis precedes cell proliferation, the relative number of medial VSMCs did not increase over 8 days, suggesting that hypertrophy alone was responsible for the increased thickness of the arterial media. These results demonstrate that MCTP causes thickening of the media of pulmonary vessels through VSMC hypertrophy and that the prolonged DNA synthesis that accompanies VSMC hypertrophy is not followed by proliferation.

Vitamin A or zymosan pretreatment attenuates alpha-naphthylisothiocyanate-induced liver injury.

alpha-Naphthylisothiocyanate (ANIT) is a cholangiolitic hepatotoxicant that causes periportal hepatic injury in the rat that is neutrophil- and platelet-dependent. Since macrophages have recently been implicated as participants in some chemically induced hepatotoxicities, we evaluated the role of these cells in ANIT-induced hepatic injury. Rats were treated with gadolinium chloride (GdCl3), an agent which decreases hepatic macrophage numbers and activity, zymosan, an agent which increases hepatic macrophage numbers, or vitamin A, which increases hepatic macrophage activity. GdCl3 did not ameliorate ANIT-induced hepatotoxicity, as demonstrated by a lack of attenuation of any of the markers of hepatic insult evaluated. In contrast, pretreatment with either zymosan or vitamin A decreased ANIT hepatotoxicity. Zymosan administration reduced blood neutrophil numbers and influx of neutrophils into the peritoneum after intraperitoneal glycogen administration but did not affect hepatic neutrophil accumulation in ANIT-treated rats. To determine if macrophages were important in the protection by vitamin A, rats were cotreated with GdCl3 and vitamin A. GdCl3 did not alter the protection from ANIT hepatotoxicity afforded by vitamin A. Vitamin A treatment decreased ANIT and glutathione concentrations in bile at 1 and 4 hr after ANIT administration but had a minimal effect on plasma ANIT concentration. In summary, pretreatment of rats with zymosan or vitamin A but not GdCl3 attenuated ANIT-induced liver injury. The protection afforded by zymosan may derive from its effects on neutrophils or platelets. The protection by vitamin A appears to result from its effect on the transport of ANIT into bile. The results suggest that hepatic macrophages are not required for the manifestation of ANIT hepatotoxicity.

Platelets and alpha-naphthylisothiocyanate-induced liver injury.

Administration of alpha-naphthylisothiocyanate (ANIT) to rats results in periportal cholangiolitic hepatopathy. Inflammation is a hallmark of the liver injury, and expression of toxicity is dependent on blood neutrophils. The role of other cellular mediators of inflammation in ANIT-induced hepatic insult is unknown. We hypothesized that platelets participate in the expression of ANIT hepatotoxicity. To test this, circulating platelets were decreased by administration of anti-rat platelet serum (PAb) prior to treatment of rats with ANIT. The PAb treatment regimen effectively reduced circulating thrombocytes over the course of the experiment. Twenty-four hours after oral ANIT administration, rats were euthanized and liver injury was estimated by increases in serum alanine aminotransferase (ALT) and gamma-glutamyltransferase (GGT) activities. Cholestasis was assessed by measurement of serum total bilirubin concentration and bile flow. Reduction in platelet numbers was associated with attenuation of the increases in plasma ALT activity and bilirubin concentration seen after ANIT administration. However, PAb treatment did not attenuate the increase in plasma GGT, a marker of biliary epithelial cell injury. ANIT-induced changes in platelet function were assessed by evaluating platelet aggregation responses in platelet-rich plasma from rats treated with ANIT in vivo. ANIT treatment modestly decreased ex vivo platelet aggregation in response to ADP and collagen stimuli. To address further the role of platelet-derived cyclooxygenase products in ANIT hepatotoxicity, rats were treated with aspirin or ibuprofen. Neither pretreatment ameliorated ANIT-induced hepatic insult. These results suggest that platelets contribute to the expression of ANIT-induced liver injury, but they do not appear to act through the production of cyclooxygenase metabolites.