MMP-2 and TIMP-1 predict healing of WTC-lung injury in New York City firefighters

The collapse of the World Trade Center (WTC) on 9/11/2001 produced a massive exposure to dust and products of combustion [1‐3]. The Fire Department of New York (FDNY) bureau of health services rapidly responded to this atrocity, initiating a medical monitoring program in October of 2001. Over 13,000 exposed rescue workers have been longitudinally followed by the FDNY-WTC-Medical Monitoring and Treatment Program. Approximately 7,000 exposed workers had serum samples drawn, stored and FEV₁ and FVC measured within six months of 9/11/2001. A vast majority of those exposed had an acute decline in lung function in the first six months followed by stabilization. There was no recovery in lung function for the group as a whole, but a minority of those exposed recovered lung function over the following six and a half years.

We used serum obtained soon after the exposure to measure serum biomarkers of lung injury during the process of disease evolution. We reported that inflammatory cytokines, lipids and other measure of metabolic syndrome as well as biomarkers of cardiovascular risk predict abnormal lung function years later [4‐6].

The balance of increased protease activity and reduced anti-protease activity are components of many diseases including cigarette-induced chronic lung disease and other causes of accelerated lung function decline [7‐10]. Genetic association studies with matrix metalloproteinases (MMPs) demonstrate a strong association with the development of lung disease [7]. MMP-1 is induced in smokers with COPD and its overexpression in mice causes emphysema [11, 12]. The destructive effects of MMPs are inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs). Since most clinical investigation focuses on disease, there is little data on the role of MMPs and TIMPs in the resistance to the damaging effect of dust exposure [13‐16]. One carefully done pathologic study demonstrated increased MMP-2 and TIMP-1 mRNA expression in surgically removed lung and predicted improved FEV₁ in COPD patients [17]. Serum MMP and TIMP expression reflects the severity of COPD supporting an investigation of a link between serum MMP/TIMP balance and lung function in the WTC exposed cohort [18].

This study investigates serum expression of MMPs and TIMPs soon after damaging particulate matter exposure and tests if protease/anti-protease balance is associated with a subgroup that demonstrates an above average healing potential after WTC-LI. We report that elevated TIMP-1 and MMP-2 predicts recovery of lung function while elevated MMP-1 reduces the odds of recovery years after WTC exposure.

We report that elevated MMP-1, MMP-2 and TIMP-1 in serum shortly after WTC exposure predicts subsequent return of FEV₁ to pre-exposure values. All subjects in this nested case control investigation had significant WTC dust exposure, arriving at the collapse site within 2 days of 9/11/2001. The FDNY measured serial FEV₁ pre and post 9/11. We focused our current study on a subgroup of highly exposed individuals who did not suffer persistent FEV₁ decline. This resistant subgroup had greater than average reduction in FEV₁ immediately after exposure but returned to pre-exposure FEV₁ over the next 6.5 years. Because serum was drawn well before the pulmonary function test that demonstrated recovery, the biomarker information reflected the evolving response to injury. MMP-2 and TIMP-1 expression above the 75^th percentile are protective biomarkers, significantly increasing the odds of resistance between 4.2 and 5.4 fold. Alternately, elevated MMP-1 is a risk factor, reducing the odds of resistance by 73%. The biomarker model using serum MMP-1, MMP-2 and TIMP-1 concentration predicted resistance with a sensitivity of 74%, a specificity of 86% and a receiver operator characteristic of 0.90.

As expected in this highly exposed group, both cases and controls suffered an acute reduction in FEV₁ as a result of WTC exposure. Resistant cases differed from controls because they returned to 99% of their pre-exposure FEV₁. Over the 6.5 years post 9/11 FEV₁ returned to only 91% of their pre-exposure FEV₁. The return of FEV₁ to pre-exposure levels provides evidence that resistant cases have above average capacity to heal after an acute injury.

Elevated TIMP-1 and MMP-2 expression increases the odds of being resistant 4.1 and 5.3 fold respectively. These results remain significant after adjusting for multiple comparisons. Although TIMP and MMP over expression have been observed in COPD and are probably affected by chronic injury secondary to cigarette smoke or other damaging processes, interpreting the cause and effect relationship between lung function and MMP/TIMP balance in humans has been challenging [22‐24]. A carefully done study of 63 patients who had surgery for lung cancer or lung transplantation demonstrated that increasing TIMP-1 and MMP-2 mRNA in the small airways and/or the parenchyma surrounding the small airway was positively associated with FEV₁[17]. Our findings on particulate matter induced lung injury are consistent with these findings in smoking related COPD patients. The mechanisms involved in the protective effects of TIMP-1 and MMP-2 in response to particulate matter induced lung injury require further study.

Since resistant cases were defined by a FEV₁ of > 107%, starting close to this threshold should increase the likelihood of crossing this defining value. After adjusting the logistic model for pre-9/11 FEV1, the association of MMP-1 MMP-2 and TIMP-1 with better than average post exposure FEV₁ markedly improved as quantified by their ORs. This suggests that while pre-existing differences in lung function is a confounder including them in the regression allowed the biomarker-lung function association to remain robust. We briefly examined if excluding individuals who failed their MCT by the 3^rd level would alter our results. We found that the ORs of MMP-1, MMP-2 and TIMP-1 remained significant and of the same magnitude (data not shown). Furthermore, we repeated the analysis in a population with their Pre-9/11 FEV₁% predicted constrained; cases n = 58 had an FEV₁ of 96–146 and controls n = 58 had an FEV₁ of 96–143. Using this constrained population OR (95% CI) were as follows: MMP-1 0.318 (0.098-1.039), MMP-2 4.993 (1.664-14.983) and TIMP-1 8.061 (2.423-26.825). Importantly, patients with resistance to WTC-LI had at subspecialty pulmonary evaluation increased TLC measured on plethysmography, increased alveolar volume measured by methane dilution and increased diffusion of carbon monoxide than controls. In animal models, MMP-2 is required for normal lung development and failure to produce MMP-2 leads to emphysema and collagen deposition around the bronchioles [25‐27]. Our data is consistent with the hypothesis that MMP-2 and TIMP-1 are biomarkers of an individual’s intrinsic capacity to heal after irritant induced lung injury.

The resistant cases also had significantly less bronchial wall thickening on chest CT. Accumulation of inflammatory cells in the bronchovascular bundle likely accounts for this radiographic finding [28]. Our Chest CT findings suggest that the resistant group accumulates fewer inflammatory cells around the bronchovascular bundle after dust exposure. Interestingly, in rodent models of lung injury, MMP-2 expression reduces bronchovascular inflammatory cells and enhances repair [29, 30]. Since the chest CT was performed years after the insult, the mechanism that produced the bronchial wall thickening persists for years after the original exposure that precipitated the inflammation.

Conversely, elevated MMP-1 reduced the odds of being resistant to WTC-LI to 0.27. This association, however, becomes insignificant when adjusted for multiple comparisons. We have maintained MMP-1 in the model because of the overwhelming evidence that this protease is an important is disease pathogenesis. Over expression of MMP-1 leads to emphysema in animal models [31]. In humans, MMP-1 is expressed in type II pneumocytes of patients with COPD but not controls [11, 12]. The 73% reduction in the odds of having above average FEV₁ is consistent with the damaging effects of this protease on lung integrity.

Since we excluded ever smokers and individuals with pre-9/11 lung disease, our results are not confounded by these common causes of FEV₁ decline that are unrelated to inhalation of WTC dust. Another advantage of this study is the SPE PFT was performed during the initial pulmonary evaluation prior to treatment initiation. Therefore our results are not confounded by treatment effect. In spite of our objective case definition, using FEV₁ as a single measure did produce misclassification of disease. The resistant group with FEV₁ > 107% at SPE did have a individuals with evidence of airway injury. Up to 22% of the resistant cases had airway reactivity on PFT or radiographic evidence of bronchial wall thickening. This misclassification of disease should bias toward the null. In spite of this bias, we observed highly significant associations between biomarkers in serum drawn soon after exposure and above average lung function years later.

This nested case control study has several limitations. The cohort was highly unusual, suffering an acute overwhelming exposure to PM that overwhelmed normal protective mechanisms. The results therefore have limited generalizability. The findings require replication in independent particulate matter exposed cohorts. Even though the serum biomarkers were expressed years before the FEV₁ that defined resistance to lung injury, the results are correlations and do not imply causation. It is possible that exposure lead to an alteration in these biomarkers due to different mechanisms controlling lung injury. Alternately, pre-existing attenuation of these biomarkers may lead to differential healing. Further, investigation in model systems and longitudinally followed cohorts is required to better understand the role, if any, of MMP-1 and TIMP-1 in healing after PM induced lung injury. Finally, this study had no unexposed control group because the few unexposed workers were markedly different from the exposed group with poor health that prevented them from working at the WTC collapse site. This restricts our ability to assess the impact of WTC exposure to the observed biomarker disease relationship.

This report documents the serum biomarkers that predict better than average FEV₁ after massive dust exposure. This group had evidence of healing with return to pre-9/11 FEV₁ after a significant drop immediately post exposure. The processes initiated by WTC exposure impacted multiple distinct injury and repair pathways. One interpretation of the findings is that biomarkers of resistance reflect biological processes leading to healing after particulate matter induced injury.