Transforming growth factor-␤ in critical illness Ruben Zamora, PhD; Yoram Vodovotz, PhD Definitions and Aliases The transforming growth factor-␤ (TGF-␤) family of three related mammalian peptides (representatives of a much larger superfamily of homologous, evolutionarily conserved proteins) exerts a multitude of effects on most cell types (1, 2). Of these, the TGF-␤1 isoform is the one most closely associated with immune modulation (3), although TGF-␤2 also participates in some aspects of immune regulation (notably in the brain) (4). The numerous biological functions of all TGF-␤s require a set of posttranslational modifications termed “activation.” The bioactive forms of the TGF-␤s are 25-kDa homodimers produced from ⵑ50kDa monomers that dimerize to form the ⵑ100-kDa TGF-␤ precursor. This dimeric precursor is cleaved intracellularly by furin proteases to yield the 25kDa active TGF-␤ dimer, which remains associated with the remaining portion of its own pro-form, the latency-associated peptide (LAP, ⵑ75 kDa). This complex is termed “latent TGF-␤”and is secreted in this form. Other proteins, such as latent TGF-␤ binding protein (LTBP, which targets TGF-␤s to the extracellular matrix) or ␣2 macroglobulin (which is associated with circulating TGF-␤1) can bind to this complex, creating the so-called large latent complex (5). Latent TGF-␤ is activated by a process that involves dissociation and degradation of LAP by proteins (e.g., plasmin and transglutaminase), heat, chaotropic agents, acid, and oxygen and nitrogen free radicals (1, 5– 8). Al-

From the Department of Surgery, University of Pittsburgh, Pittsburgh, PA. Both authors are supported by a NIAID grant. Dr. Vodovotz has consulted for Immunetrics, Inc. and holds stock and equity interest in the company. He has also received a grant from Pittsburgh Lifesciences Greenhouse to Immunetrics, Inc., and has filed a patent application for an algorithm estimating the outcome of inflammation following injury or infection. Dr. Zamora has no financial interests to disclose. Copyright © 2005 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/01.CCM.0000191725.59611.14

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though TGF-␤1 can auto-induce its own expression (1), the posttranslational control of TGF-␤1 through activation is arguably the most potent regulatory mechanism for this cytokine (5). Once activated, TGF-␤1 binds to its signaling receptor complex (type I, type II, and type III in concert). Active TGF-␤s signal via serine/threonine kinase activity of the type II receptor to downstream effectors, most commonly members of the smad family (9, 10).

History of TGF-␤ TGF-␤1 was the first member of the TGF-␤ family to be discovered, and although numerous activities later would be ascribed to this cytokine, it was initially characterized as a protein, distinct from TGF-␣, which synergized with epidermal growth factor to cause anchorage-independent growth of epithelial cells (11). Quickly thereafter, various roles were ascribed to all three mammalian members of the TGF-␤ superfamily in inflammation/immunity (3, 12), cell proliferation (13), development (14, 15), cancer (1), and a plethora of other physiologic and pathologic processes. As summarized in Table 1, numerous studies have examined the role of TGF-␤1 in both animal models of sepsis and clinical settings (discussed further below).

Assays and Caveats Various assays for TGF-␤s have been used (16). The original assay was based on the capacity to induce the anchorage-independent growth of normal rat kidney fibroblasts (11). Subsequently, two other bioassays based on cell proliferation were developed specifically for TGF-␤1, which exploited the sensitivity to TGF-␤1 of the mink lung epithelial cell line CCL-64 (17) or of the T cell clone HT-2 (18). Of note, these assays involve suppression of proliferation and may suffer from artifacts for this reason. To circumvent these issues

and to ensure specificity for TGF-␤1, a variation of the CCL-64 assay was developed in which CCL-64 cells were stably transfected with a construct coding for the TGF-␤1-sensitive plasminogen activator-inhibitor-1 (PAI-1) promoter, upstream of the luciferase reporter gene (19). It should be noted that all of these bioassays are sensitive to culture conditions; many commercially available sera contain high levels of TGF-␤1. An alternative, common, and commercially available method involves an enzyme-linked immunosorbent assay (ELISA), which detects active TGF-␤1 (or total TGF-␤1 after activation by transient acidification or treatment with urea (20). The TGF-␤s can also be detected in situ by immunocytochemistry (8, 21–24). Several important caveats should be kept in mind when assaying TGF-␤1, in addition to those mentioned above. Since the TGF-␤s are highly conserved at the protein level (only one amino acid distinguishes mouse and human TGF-␤1, for example), it is relatively straightforward to assay the protein and/or bioactivity as described above. However, there is greater divergence among species at the mRNA level. Another point that should be kept in mind is that there are settings in which the levels of TGF-␤1 protein in the culture medium will appear to be constant, and yet cell-associated TGF-␤1 will be converted from latent to active (8, 24). Accordingly, any conclusion that TGF-␤1 does not play a role in human sepsis, on the basis of findings demonstrating that TGF-␤1 levels do not change in sepsis patients (25, 26), should be interpreted cautiously. The effects of TGF-␤1 are likely to be manifest at the tissue level, where local activation (27) may play a major role. Finally, it should be noted that in many settings researchers find that TGF-␤1 mRNA levels are elevated at baseline and do not change with treatment or time; again, activation of latent TGF-␤1 protein may be a preCrit Care Med 2005 Vol. 33, No. 12 (Suppl.)

Table 1. TGF-␤ in sepsis Experimental Model/Inflammatory Condition or Disease Cecal ligation and puncture (CLP) in mice

Isolated human leukocytes isolated exposed to purified human platelet TGF-␤ 1 Rat model of septic shock produced by Salmonella typhosa lipopolysaccharide (LPS)

Rat model of Gram-negative bacterial sepsis LPS model in mice over-expressing a cDNA coding for active TGF-␤1 in the liver (Alb/TGF-␤1)

Rat model of endotoxic shock In vitro: rat vascular smooth muscle cells and mouse peritoneal macrophages Serum cytokine levels in HIV-1-infected patients with Mycobacterium avium complex (MAC) bacteremia Model of enteral ethanol delivery in CD14 knockout and wild-type BALB/c mice Rat thermal injury model

Thermally injured mice infected with Pseudomonas aeruginosa PAO1

LPS/D-galactosamine-challenged mice Serum cytokines in burned patients with and without sepsis

Plasmodium falciparum malaria in children Plasma levels of trauma patients

Plasmodium yoelii infection in mice

Streptococcus pneumoniae infection in mice Serum cytokines in patients with severe sepsis due to community-acquired infections Cytokine stimulation of primary human hepatocytes and hepatoma cell lines

Role of TGF-␤ and Specific Isoforms Elevated blood TGF-␤ value is associated with depressed splenocyte interleukin 2 release at 24 hrs after CLP TGF-␤ 1 causes early monocyte suppression of CD14 expression TGF-␤1 markedly reduces iNOS mRNA and protein levels in several organs. In anesthetized rats, after an initial 25% decrease in mean arterial pressure, TGF-␤1 arrests LPS-induced hypotension and decreases mortality. Increased circulating levels and cellassociated TGF-␤ protein levels in adherent splenic cells Reduced serum levels of the NO reaction products, reduced NOS2 protein in peritoneal exudate cells but more NOS2 mRNA and protein in both liver and kidney Alb/TGF-␤1 mice treated with LPS had eightfold higher serum tumor necrosis factor alpha (TNF-alpha) levels and experienced increased mortality TGF-␤1 reduces LPS-induced HO-1 mRNA and protein expression in heart and lung. TGF-␤1 downregulates cytokine-induced HMG-I(Y) mRNA and protein levels TGF-␤ levels are elevated consistent with advanced HIV-1 disease TGF-␤ levels are increased significantly in wild-type mice fed ethanol but not in the CD14 knockout animals Thermal injury induces a significant increase in serum TGF-␤ in burned animals compared to control animals PAO1 challenge alone or the combination of thermal injury plus PAO1 infection upregulates TGF-␤ expression in the skin and liver TGF-␤1 levels are markedly elevated in the liver challenged mice Serum TGF-␤ level is initially increased postburn in all patients. A low secondary TGF␤1 response is observed in the nonsurvivors. Plasma levels of TGF-␤ are elevated

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Lower TGF-␤1 levels are associated with liver and renal insufficiency Higher TGF-␤1 levels 6 h after ICU admission increase the risk of sepsis The virulence of malaria infection is dependent upon the cellular source of TGF␤ and the timing of its production TGF-␤ is elevated in the spleen

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Higher serum levels of TGF-␤1 as compared to healthy patients. Baseline levels of TGF␤1 are significantly higher in survivors TGF-␤1 inhibits lipopolysaccharide-binding protein (LBP) transcript accumulation and LBP protein synthesis induced by IL-6, IL1␤ and dexamethasone

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TGF, transforming growth factor; iNOS, induced nitric oxide synthase.

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Figure 1. Diverse roles of transforming growth factor (TGF)-␤1 in the sepsis. Injury and/or infection cause the release of TGF-␤1 from platelets, the largest single source in the body (1). TGF-␤1 is possibly the most potent polymorphonuclear leukocyte (PMN)/monocyte chemoattractant known and thus initially acts as a pro-inflammatory agent (3). Cytokines produced by activated PMNs and monocytes, including tumor necrosis factor (TNF), interferon (IFN)-␥, interleukin (IL)-6, and IL-10 all modulate TGF-␤1 activation and expression (12). Effector enzymes such as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the inducible nitric oxide synthase (iNOS), whose expression and/or activity is stimulated by pro-inflammatory cytokines, produce oxygen and nitrogen free radicals, respectively (12). This effect of TGF-␤1 may occur subsequent to activation of latent TGF-␤1 by either reactive oxygen (7) or nitrogen (8) species. This complex biology of TGF-␤1, including suppression of T, B, NK, and LAK cells, suggests that therapeutic uses of TGF-␤1 will require much additional study.

dominant mode of regulation of this potent cytokine.

employ such tools as the recently developed mechanistic mathematical models of acute inflammation (31, 32).

TGF-␤1 in Critical Illness Multiple studies have suggested a role for TGF-␤1 in critical illness (Table 1). Notably, TGF-␤1 markedly arrested lipopolysaccharide-induced hypotension and decreased mortality (28). In trauma patients, lower TGF-␤1 levels were associated with liver and renal insufficiency, and higher TGF-␤1 levels after admission to the intensive care unit were associated with an increased risk of sepsis (29).

CONCLUSIONS TGF-␤s helped define the concept of pluripotent cytokines, whose effects depend on local context, timing, and dose (30). Because of their diverse effects on numerous cell types, tissues, and organs, their complex regulation (5), and their importance in critical illness (Fig. 1), they constitute a fascinating area of research. In order to approach TGF-␤1 as a therapeutic target given this complex biology, however, it may be necessary to S480

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