Ex vivo cytokine production

Rapaport MH, Manji HK. The effects of lithium on ex vivo cytokine production. Biol Psychiatry 2001 50(3) 217-24. 

Tissue cross-reactivity studies are required by FDA for monoclonal antibody products to determine if the product binds to target and/or nontarget tissues. They are also performed for nonmonoclonal biopharmaceuticals if warranted. For example, ARANESP was tested in a human tissue panel ex vivo to determine if it bound to nontarget tissues or cross-reacted with related cytokine receptors. These studies are also used to explore known or potential clinical adverse safety events (i.e., mechanism of toxicity). For example, one patient in a Raptiva study developed unilateral hearing loss. This finding was further evaluated by cross-reactivity studies with human optic chiasm, acoustic nerve, and inner ear tissues. 

Six studies examined the effects of oral GLA supplementation on the ex vivo production of inflammatory cytokines by monocytes (Table VII) only two of these studies found a modest reduction in the secretion of IL-1, IL-6, or TNF (Furse et al., 2001 Watson et al., 1993), whereas the remaining four studies found no change in the secretion of these cytokines. One study that used both lipopolysaccharide (LPS) and IL-1 a to stimulate the production of IL-1 p found that GLA supplementation reduced the LPS-induced pro-IL-lp mRNA by only 17%, and it reduced the IL-la-induced pro-IL-lp mRNA by 75% (Furse et al., 2002). In this study, GLA supplementation also increased the secretion of IL-1 receptor antagonist. It is possible that some of these studies failed to detect the effect of GLA on the secretion of inflammatory cytokines simply because they did not use the appropriate stimulus. Overall, the effects of GLA on lymphocyte and monocyte functions appear much weaker compared with those of fish oils. Even if the effects of GLA are modest, it would be of great interest to determine whether GLA would enhance the antiinflammatory effects of EPA and DHA. 

Fig. 3.8 KLKL5KLK induces antigen-specific cellular type 2 responses. The protein ovalbumin (OVA) was injected twice (on days 0 and 28) alone or in combination with KLKL5KLK or alum. Splenocytes of vaccinated mice were restimulated ex vivo with OVA to determine the specific production of IL-5 (type 2 cytokine).

Additional parameters that are readily incorporated into a stand-alone immune function test such as the KLH-TDAR model include ex vivo lymphocyte proliferation, cytokine protein expression, and immunophenotype analysis any or all of which can enhance hazard identification and characterization of a potential immunotoxicant. While the KLH-TDAR is an example of a combined immune function screen and mechanistic study, the ex vivo methodologies described herein are generally applicable to toxicology studies that do not include an immunization protocol. Moreover, the methodologies are not species-specific however, responsiveness to various stimulants to induce ex vivo lymphocyte proliferation and cytokine production may differ across species and strain, requiring procedural optimization for a given species and ex vivo test. 

Lymphocyte proliferation and cytokine production form the basis of progression to an immunocompetent response, and drug-induced alterations in either or both parameters can indicate a potential for impaired host defense or selftolerance. Furthermore, ex vivo functional assays have the potential to elucidate mechanisms of drug-induced immunotoxicity and may provide essential information to address human risk (i.e., relevant host defense models). Finally, state-of-the-art methodologies enable rapid, highly sensitive and comprehensive evaluations of leukocyte activity, and implementation in preclinical evaluations may assist in the identification and/or characterization of drug-induced immunotoxicity. 

We found that treatment of mice with IL-4 or IL-10 (1 fig) significantly reduced the production of NO from peritoneal macrophages collected from lipopolysaccharide-treated animals ex vivo (Szab6 et al., 1994d Perretti et al., 1994) (Fig. 5). In this case IL-4 was more potent than IL-10, with a significant inhibition seen even at a dose as low as 0.1 fig per mouse. In cultured microvascular endothelial cells inhibition of iNOS induction by IL-10 is not seen when the cytokine is added simultaneously with the stimulus, but this is effective when a 16-hr pretreatment protocol is used (Schneemann et al., 1993). 

In a mouse vaccination model adapted to study the effect of prebiotics, the animals were vaccinated twice with the Influvac Orthomyxovirus influenza) vaccine (booster vaccination after 21 days). The response to the vaccination was measured at day 30 after the first vaccination. Parameters used to identify the response to vaccination were DTH response (skin response after local subcutaneous vaccine injection as an in vivo measure for Thl-mediated immunity), plasma titers of specific antibodies, ex vivo lymphocyte stimulation, T-cell prohferation, cytokine production, and natural killer cell activity. A specific prebiotic mixture (GOS/lcFOS) significantly stimulated the vaccination response in a dose-dependent manner and increased the DTH response indicating a modulation of the immune system toward a Thl-dominated immune response. This effect only... 

The production of inflammatory cytokines and cell adhesion molecules (CAMs) by the arterial endothelium are key cellular events involved in the development of atherosclerosis. Activation of the endothelium results in the release of vascular cytokines such as interleukin-1 (IL-la) and tumor necrosis factor alpha (TNF-a). These cytokines induce the expression of CAMs such as intracellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1), which, together with activated monocyte chemoattractant protein-1 (MCP-1), recruit monocytes through the vascular wall, where they are involved in foam cell formation. The nuclear transcription factor, NF-kB, is a mediator in TNF-a-induced expression of cell adhesion molecules and MCP. NF-kB is activated by an atherogenic diet (Liao et al. 1993) and oxidized LDL (Brand et al. 1997), and activation is inhibited by various antioxidants (Kunsch and Medford 1999). Therefore it is of particular interest that GEN attenuated NF-kB DNA binding and TNF-a release in human monocytes (Shames et al. 1999). A small, uncontrolled pilot study ( = 6) found that GEN, but not DAI, inhibited TNF-a-induced NF-kB activation in ex vivo human lymphocytes following consumption of 100 mg isoflavones/day for 3 weeks, as well as in cultured human... 

---