Interferon-gamma coordinates CCL3-mediated neutrophil recruitment in vivo

Most respiratory virus infections are relatively benign and self-limited events. However, infection with highly pathogenic viruses can result in more severe sequelae, in which disease progresses to respiratory failure due to uncontrolled inflammation, pulmonary edema, and damage to lung tissue [1‐5].

As part of an ongoing effort to understand inflammatory responses during severe respiratory virus infection, we have developed an inhalation model using the natural rodent pathogen, pneumonia virus of mice (PVM). Originally identified by Horsfall and colleagues [6, 7], PVM is a pneumovirus (family Paramyxoviridae) that is closely related to respiratory syncytial virus (RSV), and is among the few characterized mouse models of virus-induced acute respiratory distress syndrome (ARDS) [7‐9]. Among the prominent features of this infection, a minimal intranasal inoculum (30 – 100 pfu) results in robust virus replication within bronchial epithelial cells that is accompanied by profound granulocyte recruitment. In the absence of pharmacologic intervention, PVM infection progresses to pulmonary edema and respiratory compromise, similar to the more severe forms of RSV infection experienced by human infants [10, 11]. In our earlier studies, we identified the chemokine CCL3 (MIP-1α) as a crucial component of this inflammatory response. PVM not only elicits production of CCL3 by infected bronchial epithelial cells [12], mice devoid of CCL3 or its receptor, CCR1, recruit dramatically fewer neutrophils to airways [13]. Blockade of the CCL3/CCR1 proinflammatory signaling pathway in conjunction with antiviral therapy resulted in improved survival in response to an otherwise lethal virus inoculum [14, 15]. As CCL3 is only one of several major pro-inflammatory signaling pathways activated by PVM infection [12], there is certainly the possibility of additive, synergistic, or hierarchical means to promote and to amplify the ongoing inflammatory response.

Although first identified as a component of the antiviral response to Sindbis virus [16], the role of the Th1 cytokine, interferon-γ (IFNγ) in pneumovirus infection remains uncertain. IFNγ is readily detected in bronchoalveolar lavage fluid and nasal washings from RSV-infected infants [17, 18], and minimal or absent response has been correlated with poor clinical outcome [19‐24]. IFNγ is also detected in BAL fluid of BALB/c mice in response to challenge with RSV virions [25, 26] and plays a role in limiting the inflammatory response to secondary challenge and in generating the allergic histopathology in response to formalin-fixed RSV vaccine antigens and virion components [27, 28]. Likewise, local production of IFNγ is a prominent response to PVM infection [12, 29, 30], although its role in modulating the primary inflammatory response has not yet been fully explored.

In previous work, we demonstrated that the actions of the chemokine, CCL3, signaling via its receptor CCR1, were crucial for granulocyte recruitment to the lungs in response to PVM infection [13‐15]; CCL3 has also been shown to be a crucial mediator of granulocyte recruitment in mouse models of influenza [31]. Paradoxically, CCL3 gene-deletion results in augmented neutrophil and eosinophil recruitment in response to Cryptococcus neoformans infection [32]. Here we show that CCL3-mediated neutrophil recruitment depends directly on IFNγ signaling, both in the setting of acute virus infection and in response to heterologous CCL3 expression in the respiratory epithelium.

Granulocyte recruitment is a primary finding in severe respiratory virus infection; activation of granulocytes can result in the release of proinflammatory cytokines and proteolytic enzymes that can contribute to the ongoing lung damage [33‐37]. Interestingly, although neutrophils are recruited to the lung parenchyma in response to CCL3 via coordination by IFNγ, these cytokines alone clearly are not sufficient to induce the inflammatory state that ultimately promotes lung damage and respiratory failure. Thus, despite our findings demonstrating improved survival from PVM infection with CCR1 blockade [15], and those of He and colleagues [38], who likewise demonstrated that CCR1 antagonism provided protection against neutrophil-mediated lung injury in a mouse model of acute pancreatitis, the results presented here, in which we observe neutrophil recruitment but minimal clinical disease, suggest that neutrophil recruitment and neutrophil activation are to some extent distinct and discrete signaling events. It will be crucial to identify the proinflammatory mediators that activate and well as those that recruit neutrophils in order to have a complete picture of the proinflammatory state characteristic of PVM infection.

The experimental studies performed in this manuscript utilize both IFNγ and IFNγR gene-deleted mice, which are in BALB/c and C57BL/6 background strains, respectively. PVM infection has been explored systematically in several inbred strains of mice by Anh and colleagues [39] who determined that the C57BL/6 strain is somewhat more resistant to infection than BALB/c, but that both of these inbred strains can ultimately succumb to the sequelae of severe disease. We have used both of these strains extensively for our studies (reviewed in [7‐9]) and both respond to PVM infection with robust virus replication in lung tissue, granulocyte recruitment and local production of proinflammatory cytokines, including CCL3 and IFNγ; no systematic differences, other than the aforementioned susceptibility to infection, have been detected.

Both CCL3 and IFNγ have been detected in human studies and in mouse models of other severe respiratory virus infections, including avian influenza, SARS coronavirus, and human respiratory syncytial virus [17, 18, 40‐47], although the potential for interplay between these specific signaling pathways has not been considered previously. Our data suggest that that IFNγ and CCL3 signaling pathways, both crucial features of the response to pneumovirus infection, interact in a hierarchical fashion, as IFNγ does not elicit neutrophil recruitment on its own [Figure 5A], but is crucial for CCL3 to function effectively. Interactions between IFNγ and CCL3 may occur at the level of signal transduction, or via alterations to the neutrophil itself. As has been documented clearly, CCL3 can function alone to induce changes in calcium concentration and chemotactic responses in mouse neutrophils in vitro [48]. The current literature on interactions of IFNγ with granulocytes was recently reviewed [49]. Among the possibilities that may address our findings, Hansen and Finbloom [50] reported that human neutrophils express IFNγ receptors and Bonecchi and colleagues [51] have shown that human neutrophils respond to IFNγ with increased expression of a variety of mediators and receptors, including the primary CCL3 receptor, CCR1. It is unclear whether mouse neutrophils respond in a similar fashion, and whether or not these defined molecular responses take place in vivo, although we have shown here that neutrophils from IFNγR gene-deleted mice express transcripts for CCR1 and mobilize intracellular calcium in response to CCL3 when examined ex vivo. We have not yet explored the possibility that the IFNγ coordinates neutrophil recruitment in response to CCL3 in a more indirect fashion, possibly via one or more intermediary cytokines. An example of this phenomenon was reported by Khader and colleagues [52], who demonstrated that Mycobacterium tuberculosis-infected dendritic cells from IL-12p40 gene-deleted mice that were unresponsive to a CCL19 gradient were also overproducing the cytokine IL-10. Most intriguing, addition of IL-10 to wild-type dendritic cells reproduced the inhibited chemotaxis response.

Joseph B. Domachowske, M. D. is Professor of Pediatrics, Microbiology and Immunology at SUNY Upstate Medical University, Syracuse, New York. Helene F. Rosenberg, M. D. is Senior Investigator and Section Chief in the Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. Drs. Domachowske and Rosenberg have collaborated extensively, and together have developed the pneumonia virus of mice (PVM) model for the study of the sequelae severe respiratory virus infection in a rodent host.