|Molecular Vision 2006;
Received 24 August 2006 | Accepted 23 November 2006 | Published 2 December 2006
Determination of active TGF-β2 in aqueous humor prior to and following cryopreservation
(The first two authors contributed equally to this publication)
University Eye Hospital, Killianstraße 5, 79106 Freiburg, Germany
Correspondence to: Philip Maier, University of Freiburg, Department of Ophthalmology, Killianstraße 5, 79106 Freiburg, Germany; Phone: +49 761 270 4001; FAX: +49 761 270 4131; email: firstname.lastname@example.org
Transforming growth factor-β2 (TGF-β2) is one of the most important immunosuppressive cytokines in the anterior chamber of the eye. It is secreted as a complex with latency-associated peptide as an inactive precursor. Only the activated form of TGF-β2 can bind to its receptor and induce signaling. To date, the concentration of active TGF-β2 in aqueous humor was exclusively determined using samples that had been preserved at -80 °C. Quantitative measurements of the activated form directly after sampling have not yet been taken. The aim of this study was to investigate the effect of cryopreservation on the concentration of active TGF-β2 in the aqueous humor. Samples of aqueous humor were drawn from patients with either cataract or a corneal disorder for determination of TGF-β2 using a Sandwich-ELISA. In group I (n=30, patients with corneal disorders or cataract), one part of each sample was tested for active TGF-β2 directly after sampling, whereas the remaining material was stored at -80 °C for later analysis. Group II consisted of patients undergoing a simple cataract extraction (n=38), and active TGF-β2 levels were determined within 3 h after sampling. In Group III (n=34, patients with corneal disorders or cataract), active TGF-β2 was determined within 3 h after puncture, as were total TGF-β2 levels after acidic activation for each sample. The average level of active TGF-β2 in the aqueous humor of group I analyzed directly after sampling was 35±31 (median 37) pg/ml. In contrast, frozen samples from the same patients showed an average concentration of 155±103 (median 152) pg/ml. The average level of active TGF-β2 in aqueous humor of 38 cataract (group II) eyes was 40±24 (median 41) pg/ml determined within 3 h after puncture. The average level of total TGF-β2 in group III was 1,654±631 (median 1,542) pg/ml compared to 33±39 (median 28) pg/ml of active TGF-β2 determined directly after sampling, yielding a ratio of 2% of active to total TGF-β2. Levels of active TGF-β2 in aqueous humor determined directly after sampling were 4.4 fold lower than those measured in frozen samples. Thus, samples meant for determining active TGF-β2 levels should not be kept frozen.
Transforming growth factor-β2 (TGF-β2) is an approximately 25 kDa polypeptide encoded by a unique gene located on chromosome 1q41 . This dimeric peptide is ubiquitously distributed in human tissues and synthesized by many different human cells. It has been detected in tear fluid, in the vitreous, and in aqueous humor [2-4]. TGF-β2 is known to play an important role in wound healing and the production of the extracellular matrix. It inhibits cell proliferation and exerts various immunosuppressive effects. In the anterior chamber, TGF-β2 is relevant for the maintenance of an immunosuppressive climate, as it alters the activities of antigen-presenting cells, and suppresses T-cell proliferation, IFN-γ production, and the inflammatory activity of macrophages [4-6]. It is secreted as an inactive precursor (latent TGF-β2 complex, L-TGF-β2) by cell types such as corneal endothelial cells, cells of the trabecular meshwork, and the ciliary body [7,8]. This precursor (200 kDa) is complexed with latency-associated peptide (LAP) and bound to latent TGF-β binding protein (LTBP). L-TGF-β2 is not able to bind to its receptor until LAP and LTBP are removed extracellularly via proteolytic cleavage. The exact mechanisms by which latent TGF-β2 is activated physiologically are not completely understood. One model of activation has been proposed in which latent TGF-β is released from the extracellular matrix by proteases, localized to cell surfaces, and activated for example by thrombospondin-1  or specific integrins . Following this activation, TGF-β2 exerts its biological functions via binding to a membrane-bound heteromeric receptor .
In addition to TGF-β2 two further isoforms of TGF-β have been identified in mammals, TGF-β1 and TGF-β3. Both isoforms play an only minor role in immunomodulation of the anterior ocular segment .
Different levels of total TGF-β2 have been found in human aqueous humor, depending on the ocular disorders concerned [12,13]. The ratio of active to total TGF-β2 in the aqueous humor has been reported to be between 11 to 61% [14-19]. However, in those studies the concentration of active TGF-β2 was exclusively determined from samples that had been preserved at -20 °C to -80 °C (Table 1). Quantitative measurements of active TGF-β2 directly after sampling have not yet been taken. As for other cytokines such as tumor necrosis factor, interferon-alpha, interferon-gamma, interleukin-1 and interleukin-6 , freezing and thawing might have an influence on levels of active TGF-β2. Therefore, we aimed to answer the following questions by performing this study:
Does cryopreservation of human aqueous humor influence levels of active TGF-β2?
What is the normal value of active TGF-β2 in the aqueous humor of patients with cataract and otherwise healthy eyes?
What is the ratio of active to total TGF-β2 in aqueous humor when active TGF-β2 is determined directly after sampling?
Group I consisted of 30 patients with various ocular disorders leading to anterior segment surgery. Thirteen samples were drawn before penetrating keratoplasty, two were drawn during cataract extraction following penetrating keratoplasty, two before repeat keratoplasty, and one during irrigation of the anterior chamber for endothelial immune reaction. Ten patients underwent simple cataract extraction and two patients suffered from cataract and glaucoma, undergoing combined surgery. In that group (mean age 63.8 years with 53% female and 47% male patients), we analyzed active TGF-β2 levels within 3 h after sampling, and following cryopreservation of various durations.
In group II, we determined active TGF-β2 levels within 3 h after sampling in 38 patients without antiinflammatory medication and without a history of eye disease except for cataract or dry age-related macular degeneration. The mean age was 76.3 years with 61% female and 39% male patients.
Group III comprised 34 patients with either corneal disorders (11 before penetrating keratoplasty, four before repeat keratoplasty, two with cataract extraction after keratoplasty, and one with endothelial immune reaction) or cataract (14 with cataract only and 2 with cataract and glaucoma). In that group, we analyzed active TGF-β2 within 3 h after sampling, as well as total TGF-β2 levels. The mean age was 63.7 years, with 47% female and 53% male patients.
Eight of those samples provided enough material to determine active TGF-β2 directly after puncture and following cryopreservation, as well as total TGF-β2 levels (two before penetrating keratoplasty, two before repeat keratoplasty, one with cataract extraction after keratoplasty, and three with simple cataract).
All invasive procedures were performed with properly obtained written informed consent in adherence to the Declaration of Helsinki for research involving human subjects. Research was approved by our local ethics committee.
Anterior chamber puncture
Following topical anesthesia, the anterior chamber was punctured under the operation microscope within 24 h after patient referral to our institution. All eyes were rinsed with sterile solution (BSS®) prior to anterior chamber puncture. A paracentesis lancet was used to penetrate the cornea in an avascular peripheral area over a length of 1 mm. Contact with limbal or peripheral corneal vessels was completely avoided. If there was any bleeding observed, the sample was not used for further analysis. Aqueous humor (0.05-0.1 ml) was drawn into conventional tuberculine syringes without coming into contact with intraocular structures.
Determination of active TGF-β2
To compare levels of active TGF-β2 before and after cryopreservation, half of the volume was analyzed within 3 h after sampling. Between sampling and testing, we kept fresh samples at 4 °C in a refrigerator. The remaining material was stored at -80 °C for later determination. Duration of cryopreservation varied between 1 and 222 days.
The concentration of total TGF-β2 was quantified after acidic activation according to the manufacturer's manual (DuoSet Elisa Development Kit, human TGF-β2, R&D Systems, Wiesbaden, Germany).
All concentrations of TGF-β2 (active or total) were determined using a Sandwich-ELISA (DuoSet Elisa Development Kit, human TGF-β2, R&D Systems). ELISA was handled according to the manufacturer's manual except that we modified the sample volume, the concentrations of the capture, and the detection antibodies. Due to small sample volumes, 50 μl of each sample were diluted 1:2 with the reagent diluent and determined afterwards. The concentration of the capture antibody was 4 μg/ml, and of the detection antibody 200 ng/ml. The ELISA assay sensitivity was 14 pg/ml. Optical density was read using an automated platereader (GENios, Tecan, Crailsheim, Germany) with a 450 nm filter. Concentrations were calculated using Magellan software (Tecan).
Statistical evaluation was performed using SPSS 11.0 for Windows. The paired t-test and ANOVA were used, with p values <0.05 defined as statistically significant. Correlations were determined with the Pearson correlation coefficient. Concentrations below 14 pg/ml were regarded as 0 pg/ml.
The average concentration of active TGF-β2 in the aqueous humor analyzed within 3 h after the sampling was 35±31 (median 37) pg/ml with a range between <14 pg/ml and 136 pg/ml for group I (n=30). Analysis following cryopreservation of the same samples revealed an average concentration of 155±103 (median 152) pg/ml with a range between <14 pg/ml and 377 pg/ml (Figure 1). Thus, the concentration in the fresh material was statistically significantly lower than that in the frozen material (p<0.0001, t-test).
For group II (n=38), the average concentration of active TGF-β2 in aqueous humor of cataract eyes was 40±24 (median 41) pg/ml, ranging between <14 pg/ml and 116 pg/ml.
For group III, the average total TGF-β2 level was 1,654±631 (median 1,542) pg/ml, ranging between 423 pg/ml and 2,587 pg/ml. The average active TGF-β2 level of the same samples was 33±39 (median 28) pg/ml and ranged between <14 pg/ml and 136 pg/ml (Figure 2). Thus, only 2% (ranging between 0% and 20.6%) of total TGF-β2 in the aqueous humor was available in its activated form. A comparison of freshly determined active TGF-β2 in group I and III did not reveal a statistically significant difference (p=0.828, ANOVA).
We found no statistically significant correlation between the difference of active TGF-β2 levels in fresh and frozen samples and the duration of cryopreservation (r=-0.09, p=0.64, Pearson correlation, Figure 3).
Eight samples in group III raised enough material to perform three analyses (active TGF-β2 directly after sampling, active TGF-β2 following cryopreservation and total TGF-β2). The statistical analysis of those samples revealed that the level of total TGF-β2 had a markedly, but statistically not significant, effect on the magnitude of the increase in active TGF-β2 in frozen samples (n=8, r=-0.68, p=0.06, Pearson correlation, Figure 4).
We did not find any statistically significant differences between groups I and III regarding gender (p=0.623, ANOVA), age (p=0.98, ANOVA), or the underlying ocular disorder (p=0.534, ANOVA). Had the sensitivity value (14 pg/ml) of the ELISA used for these analyses been set at 14 pg/ml instead of 0 pg/ml, it would have made no statistical differences to the results we present here.
In this study, we considered whether cryopreservation of aqueous humor after anterior chamber puncture might have an influence on the level of active TGF-β2 (group I). To compare our results with those of previous studies, we determined normal values of freshly determined active TGF-β2 in cataract patients (group II). Finally, we calculated the ratio of active to total TGF-β2 (group III).
We showed that cryopreservation had a powerful effect on active TGF-β2 levels in aqueous humor, as freezing and thawing before determination substantially boosted active TGF-β2 levels. The effect observed was independent from the duration of cryopreservation. Analysis within 3 h after sampling revealed active TGF-β2 levels that were 4.4 fold lower than levels determined in frozen samples (range between 2-10 fold). Therefore, correct levels of active TGF-β2 could be identified only if samples of aqueous humor are analyzed directly after sampling and not after cryopreservation. In the determination of other cytokines (tumor necrosis factor, interferon-alpha, interferon-gamma, interleukin-1 alpha, interleukin-1 beta, and interleukin-6) from blood samples, Thavasu et al.  found that in general, the stability of cytokines in whole blood was improved by storage at 4 °C. As most biochemical processes are reduced to a minimum at 4 °C, we would not expect that a significant amount of TGF-β2 would be degraded or digested during storage at this temperature before testing. Further studies should consider whether the duration of preservation at 4 °C before analysis also influences TGF-β2 levels in aqueous humor.
Brown et al.  investigated the physicochemical activation of recombinant latent TGF-β1, TGF-β2, and TGF-β3 and reported that for human recombinant latent TGF-β1 and TGF-β2, the transition from latency occurs between pH 4.1 and 3.1, and between pH 11.0 and 11.9. Thermal activation of native and recombinant latent TGF-β1-3 occurs over the temperature ranges of 75-100 °C and temperatures above 90 °C result in thermal denaturation of TGF-β1. Lyons et al.  found that extreme pH levels (1.5 or 12) resulted in a significant activation of TGF-β, while mild acid treatment (pH 4.5) yielded only 20-30% of the competition achieved by pH 1.5. They concluded that there may be two different pools of latent TGF-β, one requiring strong acid or alkali treatment for activation and a second one that is activated by mild pH change and/or plasmin.
In our study, the increase in active TGF-β2 levels seemed to be an effect of the freezing and thawing process. Even a short period of cryopreservation (24 h) may release TGF-β2 from its association with LAP and LTBP. One explanation for this might be changes of the pH in the samples by freezing and thawing. The effective pH in aqueous environments is lowered substantially with freezing to -80 °C, especially where the protein sample is not flash frozen and the protein content is subjected to molecular crowding in the free space remaining between large ice crystals. This is easily demonstrated by placing phenol red in a container with 1-10% FBS at pH 7, freezing, and watching the dye indicator change from red to pale yellow as its environment becomes more acidic. Hence, as latent TGF-β2 is exposed to a more acidic environment, it seems probable that longer periods of storage, if not fast thawing, could induce the protein to self-cleave to the activated molecule.
Another possibility of TGF-β2 activation could be that conformational changes of latent-TGF-β2, exposing the TGF-β2 receptor binding site, are induced by freezing and thawing. Such conformational changes of proteins may be caused by crystallization as well as recrystallization, by exposure of protein molecules to the ice-liquid interface, and following interfacial tension or shear on the entrapped proteins .
The magnitude of the increase in TGF-β2 levels by cryopreservation might depend on the level of total TGF-β2 in the aqueous humor. However, we did not find a statistically significant effect. Sample size (n=8) for this analysis was small, because small sample volumes often made it difficult to perform three analyses using one sample.
The increase in active TGF-β2 levels after freezing and thawing seems to explain why the levels of active TGF-β2 determined in this study (35±31 [median 37] pg/ml) were much lower than levels reported in recent studies, in which all samples were analyzed after cryopreservation (Table 1) [14-19]. In those studies, average levels of TGF-β2 ranged from 127 to 283 pg/ml with ratios of active-to-total TGF-β2 between 11-61%. In these studies patients with various ocular disorders were included. To make our results comparable to others, we included patients with simple cataract for controls (group II) as it was done by, for example, Tripathy et al. . They found active TGF-β2 levels in cataract patients of 200±240 pg/ml (range 20-830 pg/ml) compared to 40±24 (median 41) pg/ml in our study, and Jampel et al.  found a ratio of active-to-total TGF-β2 of 61% for cataract patients. However, in cataract patients, age-related macular degeneration often coexists, and Janciauskiene et al. showed that patients with cataract and coexisting eye disease had higher levels of total protein in aqueous humor than patients with simple cataract . Further studies will establish whether this has an effect on active TGF-β2 levels as we could not perform total protein analysis from our samples due to small sample volume.
Another explanation for higher TGF-β2 levels in the cited studies might be that the investigators used different tests for determination. Bioassays were often used to determine the activated form of TGF-β2 [4,6,19,25]. However, bioassays depend on stimulation or inhibition of cell proliferation in a TGF-β-dependent manner, and are stringent in their requirements for optimal performance. Therefore, many clinical studies use ELISA techniques to determine active TGF-β2 as the level of TGF-β2 before acidic activation to establish total TGF-β2 [2,14-18]. As active TGF-β2 levels after cryopreservation in our analysis were similar to those in the cited studies, it seems unlikely that different ELISA techniques are the main reason for the increase in active TGF-β2 following cryopreservation.
Determination of active TGF-β2 levels in the aqueous humor directly after sampling is time consuming and expensive, because each sample must be analyzed within a few hours after sampling. We intend to find out whether the time period between sampling and evaluation also influences active TGF-β2 levels in further studies. However, it seems important to know exact levels of active TGF-β2 in the anterior chamber, as we found that active, but not total TGF-β2, may serve as a predictive parameter for graft rejection (unpublished data, ).
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG, RE 1382/5-1).
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